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1.
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.  相似文献   

2.
A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and reproducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.  相似文献   

3.
A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.  相似文献   

4.
Comparison of Mycoplasma hyopneumoniae strains by serologic methods   总被引：3，自引：0，他引：3  
Six field strains of Mycoplasma hyopneumoniae isolated from pneumonic lungs of pigs, reference strains 11 and J of M hyopneumoniae, Ms 42 strain of Mycoplasma flocculare, and BTS 7 strain of Mycoplasma hyorhinis were compared serologically, using hyperimmune antisera produced in rabbits. All strains of M hyopneumoniae were closely related as determined with the disk growth-inhibition test; however, differences in zone sizes indicated that some antigenic heterogeneity existed. Cross-reactions were not detected between M hyopneumoniae, M flocculare, and M hyorhinis with the growth-inhibition test. The metabolic-inhibition test was more useful for detection of intraspecies antigenic difference than was the growth-inhibition test, since antigenic diversity was clearly detected among M hyopneumoniae strains. Slight cross-reactions were observed between M hyopneumoniae and M flocculare. Using 2-dimensional immunoelectrophoresis, antigenic differences were observed among M hyopneumoniae strains, although many common components also were detected in electropherograms. Mycoplasma flocculare possessed a close antigenic relationship to M hyopneumoniae, as determined by two-dimensional immunoelectrophoresis, whereas both organisms were less related to M hyorhinis. Evidence obtained in this study indicated that strains of mycoplasmas tentatively identified as M hyopneumoniae were similar antigenically, but evidence was obtained also of some diversity in antigenic structure among these strains.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae.  相似文献   

9.
The effect of nonviable Mycoplasma hyopneumoniae on transformation of swine peripheral blood lymphocytes by mitogen was investigated. Lymphocyte transformation was evaluated as incorporation of [3H]-thymidine, using a microculture system. Mycoplasma hyopneumoniae was grown in Friis medium, inactivated with sodium azide, and washed with phosphate-buffered saline solution. Four strains of M hyopneumoniae, strain J, strain 11, and 2 low-passage isolates (1361A, 1375C), were found to suppress phytohemagglutinin-induced lymphocyte transformation. Mycoplasma hyopneumoniae strains J, 11, and 1361A reduced lymphocyte transformation by about 50%, whereas strain 1375C reduced lymphocyte transformation by 98.7%. The suppressive effect was abrogated by heating M hyopneumoniae at 60 C or at higher temperatures for 30 minutes. Sonication of the heated M hyopneumoniae cells partially restored the suppressive effect.  相似文献   

10.
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.  相似文献   

11.
The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.  相似文献   

12.
Microorganisms associated with pneumonia in slaughter weight swine.   总被引：2，自引：1，他引：1       下载免费PDF全文
The lungs of 334 pigs were obtained from two slaughter plants in Minnesota and examined in detail. Macroscopic and microscopic evaluation, direct fluorescence for Mycoplasma hyopneumoniae and bacterial culture were done on all of them and a subsample of 50 were selected for virus culture. Mycoplasma hyopneumoniae, Pasteurella multocida and Haemophilus spp. were detected in 24.0%, 34.1% and 27.0% of the lungs, commonly in conjunction with each other. One isolate of Haemophilus pleuropneumoniae serotype 2 was detected and this represents the first report of its presence in the United States. No virus was detected in any of the lungs. Lungs with both M. hyopneumoniae and Pasteurella multocida had the greatest amount of macroscopic pneumonia (9.8% of the lung). Lungs with M. hyopneumoniae or P. multocida alone had 4.9% and 5.2% of the lung involved with pneumonia respectively. Lungs with Haemophilus sp. Taxon "minor group" had 3.8% of the lung involved which was not significantly different from lungs with none of these organisms being detected (1.6%). There was a positive correlation between the extent of M. hyopneumoniae infection, as scored by FAT and the amount of macroscopic pneumonia present (r = 0.46; P less than 0.001). Likewise, there was a positive correlation between the estimated concentration of P. multocida present, as scored by the relative number of colonies on blood agar and the amount of macroscopic pneumonia present (r = 0.60; P less than 0.001). Microscopically, the amount of lymphoreticular proliferation, polymorphonuclear cells and alveolar macrophages were evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
Copper-zinc superoxide dismutase (Cu/ZnSOD), a key enzyme in defense against toxic oxygen-free radicals, is widespread in eukaryotes and several species of gram-negative bacteria. The presence of this enzyme in Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of mycoplasmal pneumonia in pigs, was examined since the polyclonal antibody against bovine Cu/ZnSOD was dominantly cross-reactive with the M. hyopneumoniae Cu/ZnSOD from whole cellular proteins. In situ activity staining on SDS-PAGE showed that the molecular mass of M. hyopneumoniae Cu/ZnSOD in reducing form was approximately 17kDa. The presence of Cu and Zn ions at the active site of the enzyme was confirmed on the basis of inhibition by KCN and by H(2)O(2). The activity of M. hyopneumoniae Cu/ZnSOD on both SDS- and native-polyacrylamide gels was completely inhibited by 2mM KCN and the gels showed no iron-containing SOD (FeSOD) or manganese-containing SOD (MnSOD) in the crude extracts. The activity of M. hyopneumoniae Cu/ZnSOD in crude extract was 70units/mg protein and was 55% inhibited by 5mM KCN and 56% inactivated by 40mM H(2)O(2). This enzyme was growth-stage dependent and evidenced markedly higher production during the early log phase. Different expression levels of Cu/ZnSOD activity in field isolates were also detected. Taken together, the presence of Cu/ZnSOD in M. hyopneumoniae was identified for the first time.  相似文献   

14.
A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.  相似文献   

15.
The type strains of Mycoplasma hyopneumoniae, M. flocculare, M. dispar, and M. ovipneumoniae, all characterized by nipple-less colonies on solid media, were compared serologically. By indirect hemagglutination and by complement fixation tests they were found to constitute a related group. By crossed immunoelectrophoresis a sharing of common antigens was demonstrated, whereas no cross reactivity was noted by the metabolism inhibition test.The type strains of Mycoplasma hyorhinis and Mycoplasma bovirhinis were included in the study for comparison. Although some cross reaction was noted, they appeared just moderately related to the nipple-less group as well as to each other.  相似文献   

16.
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.  相似文献   

17.
The chronologic localization of Mycoplasma hyopneumoniae was examined by in situ hybridization in experimentally infected pigs for a period of 35 days after intratracheal inoculation. M. hyopneumoniae DNA was detected in bronchial and bronchiolar epithelial cells from infected pigs at 7, 14, 21, and 28 days postinoculation (DPI) and in alveolar and interstitial macrophages and type I pneumocytes from infected pigs at 14, 21, 28, and 35 DPI. Strong hybridization signals for M. hyopneumoniae were detected mainly at the luminal surface of bronchial and bronchiolar lining epithelial cells. When a hybridization signal was detected at the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole also exhibited peribronchiolar lymphoid cuffing. These observations suggested that the presence of M. hyopneumoniae in different tissues could be due to a difference in the duration of the infection.  相似文献   

18.
Pathogens causing significant respiratory disease in growing pigs include Porcine reproductive and respiratory syndrome virus, Porcine circovirus 2, swine influenza virus, porcine respiratory coronavirus, Mycoplasma hyopneumoniae, and Bordetella bronchiseptica. The objective of this research was to characterize the respiratory excretion of these pathogens by acutely infected pigs. Pigs were inoculated under experimental conditions with 1 pathogen. Samples were collected from the upper respiratory tract and exhaled air. All pathogens were detected in swabs of the upper respiratory tract, but only M. hyopneumoniae and B. bronchiseptica were detected in expired air from individually sampled, acutely infected pigs. These findings suggest either that the acutely infected pigs did not aerosolize the viruses or that the quantity of virus excreted was below the detection threshold of current sampling or assay systems, or both, at the individual-pig level.  相似文献   

19.
Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection.  相似文献   

20.
Lungs from 191 slaughter pigs with gross lesions indicative of enzootic pneumonia of pigs (EPP) and 80 grossly normal lungs, all originating from 9 different herds, were subjected to microbiological and pathological examinations. The microbiological studies included both bacterial and mycoplasmal culture and also testing for Mycoplasma hyopneumoniae antigen in tissue by indirect immunofluorescent technique. M. hyopneumoniae, Pasteurella multocida and Mycoplasma hyorhinis were detected in 83%, 43% and 37% of the pneumonic lungs, respectively. Mycoplasma flocculare was the most frequently isolated organism in the non-pneumonic lungs. The greatest amounts of macroscopic pneumonia (25.2%) were recorded in lungs with all the three agents M. hyopneumoniae, P. multocida and M. hyorhinis present. The amounts of pneumonia in lungs with M. hyopneumoniae alone and in concurrence with P. multocida, were 9.3% and 15.6%, respectively. M. hyorhinis was also, in this study, associated with higher frequency of diffuse pleuritis. These findings indicate that M. hyorhinis might be involved in the pathogenesis of pneumonia in slaughter pigs. Ninety-six per cent of the isolates of P. multocida from pneumonic lungs could be characterized as type A. In the herds which had the most severe pneumonia problems, toxin production was detected in 83% of the P. multocida strains while only 28% were toxigenic in herds with subclinical to moderate pneumonia problems.  相似文献   

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