A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae.

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.     

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.     

The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells

An investigation was undertaken to assess whether polyclonal convalescent and hyperimmune sera obtained from pigs inhibit Mycoplasma hyopneumoniae induced increases in intracellular calcium [Ca2+](i) in ciliated porcine tracheal cells. Basal [Ca2+](i) in the tracheal cells was 97+/-13 nM (n=22 cells in four experiments) and after exposure to M. hyopneumoniae (300 micro g/mL or 10(11) CCU/mL), [Ca2+](i) increased by 246+/-56 nM within 100 s. After pre-treatment with hyperimmune or convalescent serum, M. hyopneumoniae increased [Ca2+](i) by 196+/-43 and 223+/-65 nM, respectively. It was found that neither hyperimmune nor convalescent serum significantly prevented the increase in [Ca2+](i) compared with M. hyopneumoniae alone. It was concluded that polyclonal antibodies produced by mycoplasma vaccination or exposure to the pathogen do not prevent M. hyopneumoniae-induced increase in [Ca2+](i).     

Experimental infection of SPF pigs with Actinobacillus pleuropneumoniae serotype 9 alone or in association with Mycoplasma hyopneumoniae

The purpose of this study was to compare in SPF pigs, the pathogenicity of an Actinobacillus pleuropneumoniae serotype 9 strain 21 (isolated from the palatine tonsils of a healthy gilt on a French nucleus pig farm, with no clinical signs or lung lesions but a highly positive reaction to A. pleuropneumoniae serotype 9 antibodies) with a pathogenic A. pleuropneumoniae strain 4915 serotype 9 (isolated in France from an outbreak of porcine pleuropneumonia). The pathogenicity of one Mycoplasma hyopneumoniae strain alone or associated with A. pleuropneumoniae strain 21 was also compared. Eight groups of 7 pigs were infected (at 6 or 10 weeks of age) and a control group was kept non-infected. Results showed that sensitivity to A. pleuropneumoniae was related to the age of the pig (6 weeks vs 10 weeks) whatever the strain. Surviving pigs infected at 6 weeks of age developed severe clinical signs, lung lesions typical of A. pleuropneumoniae and they seroconverted. In contrast, symptoms and lung lesions were almost non-existent in pigs infected with strain 21 at 10 weeks of age, but a seroconversion was observed with very high ELISA titres. These results were in accordance with those observed in the nucleus pig farm. Infection with M. hyopneumoniae alone induced typical mycoplasmal symptoms, pneumonia and seroconversion. Symptoms and lung lesions were the most noticeable in pigs infected with M. hyopneumoniae at 6 weeks of age and with A. pleuropneumoniae 4 weeks later. Our results show that the presence of A. pleuropneumoniae serotype 9 in a pig herd may be clinically unnoticed and that M. hyopneumoniae may potentiate A. pleuropneumoniae infection.     

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.     

Experimental dual infection of pigs with an H1N1 swine influenza virus (A/Sw/Hok/2/81) and Mycoplasma hyopneumoniae

Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/81) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae.     

Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

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.     

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.     

Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests.

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.     

Effect of growth in cell cultures and strain on virulence of Mycoplasma hyopneumoniae for swine

In an attempt to develop better methods for consistent induction of pneumonia in naturally born swine, using cultures of Mycoplasma hyopneumoniae, fifty 6-week-old, naturally born pigs from a respiratory disease-free herd were used in 3 trials. Pigs inoculated with Mycoplasma hyopneumoniae strain 232 (passage 21) grown for 1 passage or 5 passages in Eagle minimal essential medium plus 20% porcine serum, with or without human lung fibroblasts, had a mean (+/- SD) value range between 5.4 +/- 3.6 and 9.2 +/- 2.1% of consolidated lung area. In the second trial, pigs inoculated 1, 2, or 3 days in succession with strain 232 grown in Eagle medium or Friis mycoplasmal medium with 20% porcine serum had between 5.1 +/- 7 and 8.7 +/- 4.3% of consolidated lung area. In the third trial, virulence of Mycoplasma hyopneumoniae strains 144L (p27), 11 (p26), J (p60), and 232 (p27) grown in Friis mycoplasmal medium was compared. Pigs inoculated with those strains had 5.1 +/- 4.1, 2.6 +/- 3.1, 0, and 4.3 +/- 4% of consolidated lung area, respectively. Significant differences were not found in consolidated lung area among groups in trials 1 and 2, and among groups of pigs inoculated with M hyopneumoniae strains 144L, 11, and 232 in trial 3. Pneumonia was not detected in pigs inoculated with strain J in trial 3.     

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 nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs

A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.     

Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets

The purpose of this study was to determine the optimal route of infection and the optimal sampling sites for the recovery of M. hyopneumoniae, the etiological agent of enzootic porcine pneumonia. Virulence of two strains, BQ 14 and 116, isolated in France in 1975 and 2003, respectively, was also compared. Groups of specific pathogen free piglets were experimentally infected by the intratracheal or intranasal route. One non-inoculated pig was placed in each group of infected pigs to study direct transmission. Two groups were kept uninfected. Coughing was recorded daily. Blood samples, nasal, tonsillar and tracheal swabs and tracheobronchiolar washings were collected weekly. Pigs were killed 27-37 days post-infection. Lung lesions were scored and swabs were collected from nasal cavities, tonsils, trachea, lung, liver and spleen. All the samples, collected from live and dead pigs, were cultured for M. hyopneumoniae recovery. Results showed that both experimentally infected pigs and contact pigs developed enzootic pneumonia, whatever the route of infection and the strain tested. Direct contact transmission occurred quickly. No difference between the two routes of infection or between the two strains tested was evidenced, but high individual variations were observed between pigs. Tracheal swabs and tracheobronchiolar washings were the most effective samples to detect M. hyopneumoniae compared to nasal or tonsillar swabs. Our results also suggested that tracheobronchiolar washings could have an influence on the lesion extent observed at necropsy. M. hyopneumoniae could be re-isolated from liver and spleen of experimentally infected pigs and contact pigs.     

Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae

Cellular and humoral immune responses of pigs inoculated with Mycoplasma hyopneumoniae were investigated at postinoculation weeks (PIW) 2, 4, and 6. The response of blood lymphocytes (BL) and bronchial lymph node lymphocytes (LNL) to stimulation by M hyopneumoniae antigens was evaluated by a lymphocyte-stimulation test. Specific antibodies in serum and lung washing samples were assayed by ELISA. Immunoglobulin-positive cells in lungs and bronchial lymph nodes were identified by indirect fluorescent antibody test, using isotype-specific monoclonal antibodies. At PIW 0 to 6, BL from control and M hyopneumoniae-inoculated pigs were stimulated by M hyopneumoniae cells; however, BL from inoculated pigs generally had higher stimulation indices, especially at PIW 6. The response of LNL was influenced by previous exposure to M hyopneumoniae, as indicated by higher stimulation indices (P less than 0.01) of LNL from inoculated pigs killed at PIW 2 and 6. Specific ELISA antibodies to M hyopneumoniae in lung washings from inoculated pigs consisted mainly of IgG and IgA isotypes. Examination of lung sections by indirect immunofluorescence revealed that cells producing IgM and IgA were in controls as well as M hyopneumoniae-inoculated pigs, but IgG-positive cells were only in lungs of inoculated pigs. Resolution of pneumonia appeared to correlate with development of increased sensitization of BL, as well as development of marked increases in immunoglobulins, particularly IgG in lung washings at PIW 6.     

A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation

The sequential tissue distribution of virus was investigated using virus isolation and immunofluorescence tests in 1-day-old piglets inoculated with porcine circovirus 2 (PCV2) and/or porcine parvovirus (PPV). Enlarged mesenteric lymph nodes were seen in the pig inoculated with PCV2 alone and killed at 26 days post-inoculation (PI). One of the pigs inoculated with PCV2 and PPV and killed at 21 days PI had an enlarged liver. The pig killed at 26 days PI in this group had enlarged liver, kidneys and heart. Histopathological changes were seen in lymphoid tissues of the pigs inoculated with PCV2 alone and killed at 14 and 26 days PI. Similar, but more severe, lesions were observed in the pigs infected with PCV2 and PPV and killed from 10 days PI onwards. Histological lesions of nephritis, pneumonia and hepatitis were also apparent in these animals. Mild nephritis was also seen in the pigs infected with PPV alone and killed at 14 and 26 days PI. Moderate amounts of PPV antigen were detected in tissues from the pigs inoculated with PPV alone and killed at 14 days PI. Low levels of PCV antigen were detected, mainly in lymphoid tissues, in the pigs inoculated with PCV alone and killed at 14 days PI. Low to moderate amounts of PCV antigen were detected in a wider range of tissues in the pig in this group killed at 26 days PI. In the pigs inoculated with both viruses, PPV antigen was detected in tissues of pigs killed from 3 to 26 days PI with maximal amounts detected between 6 and 14 days PI. PCV2 antigen was detected in low to moderate amounts in the tissues of pigs killed at 14 days PI. Large amounts of PCV2 antigen were detected in most of the tissues from pigs in this group killed between 17 and 26 days PI. Virus isolation results for PCV2 generally correlated well with the results for immunofluorescent staining. PPV was isolated from almost all tissues from pigs inoculated with PCV2 and PPV, a much higher incidence of positive tissues than observed for immunofluorescent staining.     

A COMPLEMENT-FIXATION TEST FOR ENZOOTIC PNEUMONIA OF PIGS USING A COMPLEMENT DILUTION METHOD

Complement-fixing antibody to Mycoplasma hyopneumoniae in the serums of pigs experimentally infected with enzootic pneumonia was demonstrated by comparing the haemolytic titre of guinea-pig complement titrated in the presence of heated test serum, M. hyopneumoniae antigen and unheated normal pig serum with the titre obtained when the antigen was omitted. The haemolytic titres against sensitised sheep erythrocytes were determined after a fixation period of 16 to 18 hours at 5°C. When serums, collected at intervals of 3 to 7 days, from 43 pigs exposed to pigs experimentally infected with enzootic pneumonia were tested, 4.6 or more complement units were first fixed 14 to 44 (mean 23.4) days after contact began. Serums collected subsequently fixed from 4.6 to more than 31 complement units. This positive reaction usually persisted until the pigs were killed 4 to 35 weeks after contact began. Thirty-three had gross enzootic pneumonia lesions and 9 had lung lesions detected microscopically. Serum antibody was not detected in 73 weaned pigs aged 7 weeks in a pneumonia-free herd but serums from 9 of 15 unweaned piglets aged 9 to 14 days in the same herd, fixed between 3 and 7 complement units.     

Protective effects of an oral microencapsulated Mycoplasma hyopneumoniae vaccine against experimental infection in pigs.

Oral microencapsulated Mycoplasma hyopneumoniae vaccines were tested for their ability to prevent mycoplasmal pneumonia in pigs. Eighteen four-week-old specific pathogen free pigs were divided into six groups of three. Each pig of groups 1 to 4 was inoculated intramuscularly with formalin-inactivated M hyopneumoniae in adjuvant and boosted orally 14 days later with four different microencapsulated vaccine microspheres. Group 5 was used as a positive control. All 15 pigs of groups 1 to 5 were challenged at 28 days after the first vaccination by an intratracheal inoculation of pneumonic lung suspension. The three pigs of group 6 were used as a negative control. All four vaccinated groups showed some protection when challenged, but the protection was more solid in pigs boosted with vaccine D (group 4) which contained less porcine serum in the microsphere. The study indicates that oral vaccination with M hyopneumoniae could play a role in prevention and eradication of mycoplasmal pneumonia in the pig.     

猪肺炎支原体乳酸脱氢酶基因的克隆、表达、纯化及其间接ELISA检测方法的建立

根据GenBank中的猪肺炎支原体乳酸脱氢酶（LDH）基因序列设计1对引物，PCR扩增LDH基因，首先将扩增片段连接到克隆载体pMD18-T上，然后连接到表达载体pGEX—KG上，经测序正确后诱导表达。重组质粒在大肠杆菌中表达的目的蛋白以可溶性蛋白和包涵体2种形式存在。将超声波破碎的诱导菌液高速离心，其上清用Glutathione Sepharose4Bbead亲和层析纯化。用猪肺炎支原体的标准阳性血清对纯化蛋白作Western—blot检测，出现目的条带；以纯化蛋白为抗原建立了检测猪肺炎支原体抗体的间接ELISA方法，该方法具有较好的稳定性和重复性，较高的特异性与敏感性。用建立的ELISA方法与中国兽医药品监察所研制的IHA试荆盒同时检测120份临床血清，二者总符合率为92．5％。用建立的ELISA方法检测了671份临床送检不同年龄阶段的猪血清，结果显示断奶前仔猪猪肺炎支原体（Mycoplasma hyopnenmoniae，MHP）感染率为44．3％，保育猪为3．0％，育肥猪为17．44％，种猪为73．41％，这初步反映了猪喘气病在各个年龄阶段的感染率。     

Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus

The objective of this study was to determine whether vaccination with bacterins commonly used in the USA, when administered at a time typical of US protocol, enhances porcine circovirus type 2 (PCV2) replication and the incidence and severity of clinical signs and lesions characteristic of postweaning multisystemic wasting syndrome (PMWS) in conventional pigs. Sixty-one pigs free of PCV2 were randomly assigned to four groups. Groups 1 (n = 15) and 2 (n = 15) pigs served as sham-inoculated negative controls. Groups 3 (n = 14) and 4 (n = 17) pigs were inoculated intralymphoid with PCV2 field isolate ISU-40895. Pigs in groups 2 and 4 were vaccinated with Actinobacillus pleuropneumoniae (APP) and Mycoplasma hyopneumoniae (M. hyopneumoniae) bacterins 21 days before and again 1 day before inoculation with PCV2. Mild transient respiratory disease and diarrhea were observed from 13 to 34 days postinoculation (DPI) in pigs in groups 3 and 4. Half the pigs from each group were necropsied at 22 and 34 DPI, respectively. Moderately enlarged, tan-colored lymph nodes were observed in the majority of pigs in groups 3 and 4. There was a significantly (P < 0.05) longer length of viremia (2.14 +/- 0.26 versus 4.44 +/- 0.23 weeks), a higher copy number of the PCV2 genome in serum, a wider range of tissue distribution of PCV2 antigen, and an increased severity of lymphoid depletion in pigs vaccinated with commercial APP and M. hyopneumoniae vaccines and inoculated with PCV2 compared with PCV2-inoculated unvaccinated pigs. Swine producers and veterinarians may need to consider changes in vaccination protocols in herds with recurrent PCV2-associated PMWS.     

Protective effect of vaccination with culture supernate of M. hyopneumoniae against experimental infection in pigs

The protective activity of Mycoplasma hyopneumoniae inactivated vaccine prepared from sedimented whole cells and cell-free culture supernates was evaluated experimentally using hysterectomy-produced, colostrum-deprived pigs in which mycoplasmal pneumonia had been induced. The culture supernate vaccine containing less than 10(1) colour-changing units (CCU)/0.2 ml of M. hyopneumoniae significantly (P < 0.05) reduced the percentage of lung lesions compared to controls (3.2 +/- 3.9 vs. 12.2 +/- 2.2%), whereas the sedimented whole cells vaccine containing 10(10) CCU/0.2 ml of organisms provided variable protection (18.7 +/- 16.5 vs. 12.2 +/- 2.2%). Serum from the pigs vaccinated with culture supernate reacted with six protein bands of 97, 89, 65, 46, 42 and 41 kDa by immunoblot analysis. From these results, we conclude that vaccination with culture supernate of M. hyopneumoniae can provide protection against M. hyopneumoniae infection and that these antigens in the culture supernate may be closely related to the reduction of lung lesions.