The nature and detection of mycoplasmal immunogens.

Mycoplasmal infections are important causes of disease in cattle, swine, sheep, goats and poultry. Vaccination has been shown experimentally to induce protection against challenge with several mycoplasmas, and vaccines have been used to control naturally occurring mycoplasmal disease in swine, sheep, goats and poultry. Immune responses to mycoplasmal immunogens have been determined using ELISA and immunoblotting as well as other serologic techniques. However, the importance of specific immunogens as virulence factors or putative protective immunogens has generally not been determined. Investigations are underway to determine the pathogenic mechanisms and identify important virulence factors involved in mycoplasmal disease. Examples are discussed of investigations with Mycoplasma hyopneumoniae from our own laboratory. We have utilized ATP luminometry in attempts to develop better methods for quantitation of growth of M. hypopneumoniae and competitive ELISA as a potential method for in vitro quantitation of specific important immunogens.

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.

Improved protection of swine from pleuropneumonia by vaccination with proteinase K-treated outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae.

The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.

A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas.

An in vitro culture system for swine tracheal epithelial cells was developed to study the adherence of swine mycoplasmas. Swine tracheal epithelial cells were isolated by enzymatic digestion and cultured on microporous membranes. Growth medium was placed under the membrane support to create air-liquid interface feeding resulting in the cells growing cilia and microvilli on the apical surface. Two strains of Mycoplasma hyopneumoniae (pathogenic strain 91-3 and non-pathogenic type strain J) and two strains of Mycoplasma flocculare (type strain Ms42 and field isolate 7160T) were used in this study. The morphology of the cultured tracheal cells was evaluated by transmission electron microscopy. Adherence of M. hyopneumoniae and M. flocculare and damage to the cilia were demonstrated using scanning electron microscopy. The pathogenic M. hyopneumoniae strain 91-3 adhered to cilia inducing obvious damage. The non-pathogenic M. hyopneumoniae strain J did not adhere to mature cilia. Both M. flocculare strains Ms42 and 7160T adhered to mature and budding cilia. No obvious ciliary damage was observed with strain Ms42. Minimal damage consisting of a slight tangling of the cilia occurred after adherence by strain 7160T. This model will enable us to further study the role of adherence of mycoplasmas on the pathogenesis of swine pneumonia.

Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model.

An in vitro culture system was developed to investigate the induction of proinflammatory cytokines by Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). M. hyopneumoniae infected porcine tracheal ring explants were co-cultured with PRRSV infected pulmonary alveolar macrophages (PAMs) for 24h to assess the cytokine production of each pathogen alone and the interaction between the two pathogens in vitro. Semiquantitative RT-PCR was used to measure interleukin (IL) 1alpha, IL1beta, IL6, IL8, IL10, IL12 and tumor necrosis factor (TNF) alpha mRNA in PAMs. Commercial ELISAs were used to measure soluble IL1beta, IL8, IL10 and TNF in the culture supernatant. In the dual infected group, mRNA expression of IL1alpha, IL1beta, IL8 and TNF was increased. Both the M. hyopneumoniae- and PRRSV-infected only groups tended to have increased expression of IL1alpha, IL1beta and IL8 mRNA, although no statistical difference was observed. Increased levels of IL1beta, IL8 and IL10 were present in the supernatant of the dual infected group as measured by ELISA. No increase in soluble TNF was observed in any of the groups. IL8 levels appeared high in all groups independent of infection status. The cause of the elevated IL8 was unknown, however, it may have been a non-specific response by the cells to tissue damage during the harvesting of the tracheal rings. Correlation between mRNA expression and the soluble cytokine levels were similar in the dual infected groups with the exception of IL10 and TNF. Levels of mRNA and soluble protein levels in the single pathogen infected groups were not as consistent. The increased production of proinflammatory cytokines IL1alpha, IL1beta, IL8 and TNF in the group infected with both M. hyopneumoniae and PRRSV suggests that cytokine induced inflammation may play an important role in the severe, chronic pneumonia induced by the concurrent infection of the two pathogens.

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.

Prevalence of antibodies to Mycoplasma hyopneumoniae in Iowa swine.

The prevalence of complement-fixing antibodies to Mycoplasma hyopneumoniae was determined in 7,321 sera collected from breeding swine of various ages. Samples were selected randomly in approximate proportion to the number of hogs marketed annually from each of 9 crop-reporting areas in Iowa. Testing was accomplished by means of a Microtiter complement-fixation test. Of the 7,321 sera, 22% had antibody titers of 1:4 or greater to M hyopneumoniae. Of the 597 herds sampled, 60% or 357 had at least 1 animal with a titer of 1:4 or greater. Use of the chi-square association test indicated that animals which were M hyopneumoniae-positive were more often Haemophilus pleuropneumoniae-positive than those that were M hyopneumoniae-negative.

Characteristics of protective activity of Mycoplasma hyopneumoniae vaccine.

Protective activity of Mycoplasma hyopneumoniae vaccines prepared from whole cells or crude extracts was evaluated in an experimentally induced mycoplasmal pneumonia swine model. Swine were obtained at 7 to 10 weeks of age from a surgically derived herd free of porcine mycoplasmas. Vaccines prepared from whole cells, freeze-thaw-saline solution extract, supernate of culture, or saline wash of cells were treated with 0.15% formalin, incorporated with Freund incomplete adjuvant, and administered IM in 2 doses. Twenty-five to 42 days after the 1st dose of vaccine was given, pigs were challenge exposed intratracheally with supernate of lung homogenate containing only M hyopneumoniae. Necropsies were done 27 to 38 days after challenge exposure. For evaluation of protective efficacy of vaccines, determination of proportion of lung with gross lesions gave better results than histologic, fluorescent antibody or mycoplasmal isolation procedures. Whole cell vaccine containing 10(9) color-changing units of M hyopneumoniae, an amount attainable in log-phase cultures, protected swine against development of pneumonia. Whole cells, supernate of culture, and saline wash of cells induced comparable protection. Protective activity of whole cell vaccine resisted heating at 80 C. Extracts prepared according to a freeze-thaw procedure were variable in protective activity and, in some instances, enhanced lesion development.

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.

Hemagglutination and hemagglutination inhibition of turkey red blood cells with Mycoplasma hyopneumoniae.

The ability of Mycoplasma hyopneumoniae to agglutinate RBC was evaluated to develop an in vitro cytadsorption assay. Using swine RBC in a microtitration hemagglutination test, no agglutination or partial agglutination was detected. Comparison of RBC from various other species indicated that improved hemagglutination was obtained with RBC from turkeys. This hemagglutination was detected only when mycoplasma cells used in the assay had been frozen and thawed, heated at 50 C for 30 minutes, or treated with trypsin. Treatment of RBC with trypsin or neuraminidase enhanced hemagglutination. Possible surface lectin activity in M hyopneumoniae was evaluated by use of carbohydrates in a blocking assay; hemagglutination was not inhibited by any of 13 carbohydrates evaluated. Mycoplasma hyopneumoniae convalescent porcine serum and monoclonal antibodies against 2 M hyopneumoniae immunogens of molecular weights of 64,000 and 41,000 inhibited hemagglutination.

Differentiation of Mycoplasma hyopneumoniae, M flocculare, and M hyorhinis on the basis of amplification of a 16S rRNA gene sequence.

To differentiate Mycoplasma hyopneumoniae, the cause of mycoplasmal pneumonia in pigs, from M flocculare and M hyorhinis, an assay, using the polymerase chain reaction to amplify a segment of the 16S rRNA gene sequence, was developed. The assay was found to be useful for identification of field isolates, as well as for identification of laboratory-adapted strains. Amplification of DNA from M hyopneumoniae and M flocculare resulted in products of 200 and 400 base pairs, respectively. The DNA from M hyorhinis was not amplified. The assay was sensitive enough to detect as little as 1,000 genome equivalents of M hyopneumoniae and M flocculare DNA. Sensitivity was increased 100-fold by increasing the concentration of magnesium ion in the reaction buffer from 2 to 4 mM; however, DNA from M hyorhinis was also amplified under these conditions. The DNA from several walled bacteria and from other mycoplasmas was also tested, but none of these DNA samples was amplified, suggesting that the assay was specific for porcine mycoplasmas.

Prevalence of antibodies to Haemophilus pleuropneumoniae in Iowa swine.

The prevalence of complement-fixing (CF) antibodies to Haemophilus pleuropneumoniae in Iowa swine was determined by testing samples collected randomly from each of 9 crop-reporting areas (9 to 12 counties) in Iowa in approximate proportion to the number of swine marketed annually from each area. For testing, a pooled antigen composed of serotypes 1 to 5 of H pleuropneumoniae was used in a modified direct Microtiter complement-fixation test. Of 7,348 sera tested, 2,362 or 32.1% had CF antibodies (titer greater than or equal to 1:4) to the organism. On a herd basis, 417 of 597 herds or 68.8% had at least 1 animal with CF antibodies (titer greater than or equal to 1:4) to the organism. The clinical problem with H pleuropneumoniae has become significant in recent years, but by no means is the overt disease as prevalent as animals or herds with CF antibodies to the organism.

Susceptibility of sows to experimentally induced Escherichia coli mastitis.

A study was conducted to compare susceptibility of sows from 2 herds to experimentally induced Escherichia coli mastitis. Four sows each from herds R and S were inoculated intramammarily at postpartum hour 8 with a strain of E coli shown previously to be capable of producing mastitis. After inoculation with E coli, sows from herd S had higher temperatures, lower WBC counts, and lower plasma protein:fibrinogen ratios than did sows from herd R. Inoculated sows from herd S lost 83% of newborn pigs due to starvation by 14 days after inoculation, whereas sows from herd R lost none. Control, noninoculated sows from both herds had normal temperatures, normal hematologic values, and minimal mortality of piglets. Levels of antibodies (complement fixing, enzyme-linked immunosorbent assay, and agglutinating) to E coli in preinoculation sera from the 2 populations of sows did not differ. Assay of lactoferrin by radial immunoassay revealed comparable concentrations in milk of sows from both herds during the first 24 hours after sows had delivered, but significantly higher values were detected in milk from sows of herd S at postpartum days 2 and 3. The basis for the marked difference in susceptibility to E coli-induced mastitis was not determined except that "susceptible" sows (herd S) were from a conventional herd and "resistant" sows (herd R) were from a specific-pathogen-free herd.

Evaluation of the ability of tilmicosin to prevent adherence of Mycoplasma hyopneumoniae to cilia using a differentiated swine respiratory epithelial culture system.

Mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae is a serious economic problem for swine producers in the United States. Mycoplasma hyopneumoniae colonizes ciliated respiratory epithelial cells. The organism has been shown to be sensitive to tilmicosin, a synthetic macrolide, in antibiotic sensitivity assays. The efficacy of tilmicosin to inhibit adherence of M. hyopneumoniae to ciliated epithelial cells without direct contact between the antibiotic and the organism was evaluated using in vitro methods. The study demonstrated that tilmicosin inhibited the adherence of M. hyopneumoniae at the highest level tested in the system (2 microg/ml) suggesting that tilmicosin may be efficacious against mycoplasmal pneumonia.

[Experience of anesthesia during transthoracic endoscopic sympathectomy for palmar hyperhidrosis: comparison between double-lumen endobronchial tube ventilation and laryngeal mask ventilation].

In the past year we had 36 patients operated for transthoracic endoscopic sympathectomy to treat palmar hyperhidrosis. The first group composed of 17 patients receiving anesthesia with double-lumen endobronchial-tube ventilation from July-92 to April-93, and the second group composed of 19 patients receiving anesthesia with laryngeal mask ventilation from April-93 to August-93. During right lung collapse for sympathectomy, the first group patients' SaO2 (oxygen saturation) decreased from 99.65 +/- 0.62 mmHg (pre-operation) to 95.12 +/- 5.48 mmHg (at cauterization), 95.24 +/- 5.41 mmHg (5 minutes after cauterization) and resumed 99.53 +/- 0.62 mmHg after the procedure completed. During left lung collapse for left side sympathectomy, the same group patients' SaO2 decreased from 99.59 +/- 0.62 mmHg to 97.35 +/- 3.06 mmHg, 97.82 +/- 2.53 mmHg and resumed 99.65 +/- 0.49 mmHg respectively. The second group using laryngeal mask ventilation had SaO2 changes during right side sympathectomy from 99.68 +/- 0.58 mmHg (pre-cauterization) to 99.74 +/- 0.45 mmHg (when cauterization), 99.79 +/- 0.42 mmHg (5 minutes after cauterization) and resumed 99.84 +/- 0.37 mmHg after the procedure completed. During left side sympathectomy the second group patients' SaO2 changed from 99.84 +/- 0.39 mmHg to 99.42 +/- 1.50 mmHg, 99.47 +/- 1.46 mmHg and resumed 99.74 +/- 0.59 mmHg respectively. After 2-Way ANOVA with repeated measures of the SaO2 value, we could see that no matter what side operation, there were differences existed between these two groups (< 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Enthalpies of Dilution of Aqueous Electrolytes: Sulfuric Acid, Hydrochloric Acid, and Lithium Chloride.

Calorimetric measurements at 25 °C of the enthalpies of dilution of aqueous H2SO4 (0.00090 to 6.4 mol · kg-1), LiCl (0.026 to 6.7 mol · kg-1), and HCl (0.018 to 1.6 mol · kg-1) have been performed using two different isothermal calorimeters. The results of this work and that of three earlier calorimetric investigations and one Raman spectral investigation have been used to calculate values of the relative apparent molal enthalpies, and relative partial molal enthalpies for these electrolytes.

Characterization of Haemophilus spp. isolated from healthy swine and evaluation of cross-reactivity of complement-fixing antibodies to Haemophilus pleuropneumoniae and Haemophilus taxon "minor group".

Of 30 sows from a herd believed to be free of Haemophilus pleuropneumoniae infection, 2 had complement-fixing antibodies to H. pleuropneumoniae serotype 5. Necropsy and microbiological examination of the two sows revealed no evidence of H. pleuropneumoniae infection; however, Haemophilus taxon "minor group" and a urease-negative, indole-positive Haemophilus sp. were isolated from numerous respiratory tract sites in both sows. Isolation of these Haemophilus spp. was facilitated by serially diluting specimens in two broth media. Pigs from a closed, respiratory disease-free herd were inoculated with four strains of Haemophilus taxon "minor group" to determine whether the organism induces antibodies which cross-react with H. pleuropneumoniae in the complement fixation test. Antigenic heterogeneity among the taxon "minor group" strains was apparent; however, antibodies cross-reacting between these strains and H. pleuropneumoniae serotypes 1 through 5 and 7 were not detected.

Microtiter plate adherence assay and receptor analogs for Mycoplasma hyopneumoniae.

A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by use of purified swine tracheal cilia which contained receptors for the mycoplasmas. M. hyopneumoniae bound specifically to plates coated with solubilized cilia. The binding was dependent on both the concentration of cilia and the number of mycoplasmas. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited the binding of the mycoplasmas. The six inhibitors also disrupted the adherence of the mycoplasmas to intact ciliated cells. Preincubation with either mycoplasmas or cilia indicated that heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesive molecules on the surface of the mycoplasmas, while laminin blocked the receptors in cilia. The basis for the inhibition induced by dextran sulfate was unknown. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas, whereas treatment of cilia with sodium metaperiodate decreased binding. These results indicate that receptors for M. hyopneumoniae in the ciliated epithelium of the respiratory tract of pigs are glycoconjugate in nature.

Identification and characterization of a Mycoplasma hyopneumoniae adhesin.

An adhesin of Mycoplasma hyopneumoniae was identified and characterized in this study. A monoclonal antibody (MAb), F2G5, and its F(ab')2 fragments inhibited the adherence of M. hyopneumoniae to porcine tracheal cilia, the natural targets to which the mycoplasma binds during infection. MAb F2G5 detected multiple bands, but predominantly recognized a 97-kDa (P97) protein of M. hyopneumoniae on immunoblots. Affinity chromatography, conducted with immobilized MAb F2G5, mainly purified P97. The purified proteins were able to bind to cilia and blocked the adherence of intact M. hyopneumoniae cells to cilia. Immunolabeling of mycoplasmas with MAb F2G5 under electron microscopy demonstrated that the proteins recognized by MAb F2G5 were located at the surface of the mycoplasma, predominantly on a surface fuzzy layer. These results indicate that P97 functions as an adhesin of M. hyopneumoniae. The N-terminal amino acid sequence of P97 did not have significant homology with any known bacterial or mycoplasmal adhesins, suggesting that P97 is a novel protein. The predominant proteins detected by MAb F2G5 in different strains varied in size, indicating that the antigen bearing the epitope for MAb F2G5 undergo intraspecies size variation. Antigenic variation of adhesins may be a pathogenic mechanism utilized by M. hyopneumoniae to evade the porcine immune system.