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.

Antibody response of swine experimentally infected with Mycoplasma hyosynoviae.

Antibody responses of swine inoculated intranasally with M. hyosynoviae were determined using complement-fixation, latex-agglutination, metabolic-inhibition, and mycoplasmacidal tests. The infected swine developed latex-agglutinating antibodies by 6 days postinoculation, complement-fixing and metabolic-inhibiting antibodies by 9-12 days, and mycoplasmacidal antibodies which were first detected from 12 days to 8 weeks postinoculation. Antibody titers persisted for as long as 6 months postinoculation. Complement-fixing and mycoplasmacidal antibodies were mainly IgG, and latex-agglutinating antibodies were IgM. Early metabolic-inhibiting antibodies were IgM while later antibodies were mainly IgG. None of the pigs had detectable complement-fixing antibodies to Mycoplasma hyorhinis.

Adaptation of the Sensititre broth microdilution technique to antimicrobial susceptibility testing of Mycoplasma hyopneumoniae.

A broth microdilution technique is described for determining the antimicrobial susceptibility of Mycoplasma hyopneumoniae, using commercially prepared Sensititre plates. Twenty-five field isolates and two reference strains (J & 232), were tested against seven antimicrobials. Field isolates were tested in duplicate and reference strains, four times to estimate reproducibility. Ninety-seven percent of the duplicate MIC results for the field isolates were in agreement, or within one log2 dilution. Similar results were obtained with the reference strains. The isolates were susceptible to lincomycin-spectinomycin, tylosin and oxytetracycline or resistant to amoxycillin, apramycin and erythromycin. Susceptibility to furaltadone varied. This method retains the accuracy and reproducibility of broth MIC determinations, while avoiding the lengthy preparation of antimicrobial dilutions normally associated with more traditional methods.

Isolation of Mycoplasma salivarium from swine.

Mycoplasma salivarium, a common human oropharyngeal mycoplasma, was isolated from nasal and pharyngeal secretions of 14 of 284 swine in a barrier-maintained, disease-free herd. M. salivarium was recovered from one boar 6 times over a 26-month period and one time only from 13 other swine. Human isolates of M. salivarium were compared with the swine isolates by DNA-DNA hybridization and SDS-PAGE of the cell proteins and the strains were shown to be closely related. One of eight of the swine from which M. salivarium was isolated had complement-fixing antibodies and another culture-positive animal had metabolic-inhibiting antibodies to M. salivarium. Overt disease was not associated with the organism. These results support previous findings that mycoplasmas closely related to M. salivarium may be isolated from the nasopharynges of swine and they further indicate that the organism can establish persistently in swine without evidence of overt disease.

Comparison of seven isolates of Mycoplasma meleagridis.

A comparative study of seven isolates of Mycoplasma meleagridis indicated that they were indistinguishable morphologically. Two isolates, E2 and 8M92, induced hemagglutination of red blood cells of several different species while the others did not. Metabolic inhibition, growth inhibition and growth precipitation tests revealed minor differences among the seven isolates. According to these differences, isolates were divided into three groups: antiserum-sensitive isolate 1466, less sensitive isolates N, 8M92, RY3, 529 and E2 and insensitive isolate 1940. One dimensional polyacrylamide gel electrophoresis of cell proteins revealed that all isolates of M. meleagridis had virtually identical patterns and that they were electrophoretically distinct from Mycoplasma gallisepticum and Mycoplasma synoviae. When nonhemagglutinating isolate N, and hemagglutinating isolate E2 were examined by simple immunoelectrophoresis, no differences were detected. However, minor antigenic differences were detected between the two strains by means of two dimensional immunoelectrophoresis.

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.

Adherence of Mycoplasma hyopneumoniae to porcine ciliated respiratory tract cells.

Adherence of Mycoplasma hyopneumoniae to the mucosa of the distal portion of the respiratory tract of swine is an important initial event in development of mycoplasmal pneumonia. A suitable in vitro model of adherence would be useful for investigation of mycoplasmal and host cell factors involved in this process. We have developed an adherence assay, using suspensions of porcine respiratory tract ciliated epithelial cells and M hyopneumoniae. Tracheal epithelial cells, collected by use of cytologic brushes, were mixed with broth cultures of M hyopneumoniae and the mixtures were incubated, diluted, vortexed, and sedimented. Pellets were spread on glass slides, stained with a fluorescent antibody against M hyopneumoniae, and evaluated by fluorescent microscopy. Fluorescence was observed principally among cilia on the ciliated tufts of epithelial cells. Only a few organisms were observed adhering on the nonciliated parts of ciliated cells or on other cell types. When mycoplasmas were preincubated with low dilutions of serum from swine convalescing from M hyopneumoniae disease, attachment was partially inhibited (P < 0.05). Significant inhibition of attachment was not observed when organisms were preincubated with higher dilutions of convalescent serum, with purified IgG from hyperimmune serum against M hyopneumoniae, or with low dilutions of lung lavage fluids (from convalescent swine) that contained specific IgA antibodies against M hyopneumoniae. Preincubation of the organisms with periodate and trypsin abolished attachment and formaldehyde decreased it (P < 0.05), whereas a variety of carbohydrates had no effect on attachment. Preincubation with dextran sulfate, ammonium sulfate, magnesium sulfate, and methionine reduced attachment (P < 0.05). Treatment of cell-Mycoplasma mixtures with the hydrophobic bond-breaking agent tetramethylurea, or incubation in absence of salt, or at low temperature also reduced attachment (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of Mycoplasma hyopneumoniae membranes with porcine lymphocytes.

Nonspecific mitogenicity of Mycoplasma hyopneumoniae membranes for blood lymphocytes (BL) and bronchial lymph node lymphocytes (LNL) from swine was investigated. Additionally, the influence of respiratory tract exposure to the same membrane preparation on responsiveness of these cells was evaluated. Membranes utilized in lymphocyte transformation tests and for inoculation of swine were prepared by osmotic lysis of mycoplasma cells. Conventionally reared and cesarean-derived, colostrum-deprived swine were given membranes intratracheally and responses of BL and LNL to membranes were assessed from postinoculation day 0 to 14. Utilizing a stimulation index of 3 as the cutoff, heated (56 C) M hyopneumoniae membranes exerted moderate nonspecific stimulation of BL 11 of 12 times when BL were collected from normal (control or preinoculation) swine. Similarly, LNL from conventionally reared and control groups of swine were stimulated nonspecifically 4 of 6 times by the same membrane preparations. Exposure of the respiratory tract to membranes appeared to have no influence on stimulation responses of BL at postinoculation days 6 or 13, whereas moderate to marked increases in responsiveness of LNL were detected when collected at necropsy on postinoculation days 7 or 14. These findings indicated that compartmentalization of lymphocyte sensitization in the bronchial lymph nodes resulted from respiratory tract exposure to mycoplasmal membranes. Results obtained confirm that M hyopneumoniae has a moderate nonspecific stimulatory effect on porcine lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Morphologic features and hydrophobicity of the cell surface of Mycoplasma hyopneumoniae.

Cell surface hydrophobicity of Mycoplasma hyopneumoniae was evaluated by phase partitioning in a hydrocarbon-aqueous mixture, by hydrophobic interaction chromatography, and by salting out with ammonium sulfate. Results obtained by use of these techniques gave evidence that the cell surface of M hyopneumoniae is weakly hydrophobic, compared with strongly hydrophobic Staphylococcus aureus Cowan I and hydrophilic Klebsiella pneumoniae. After treatment of the organisms with trypsin, M hyopneumoniae became less hydrophobic as measured by hydrophobic interaction chromatography. Significant changes in hydrophobicity were not seen after periodate treatment. Electron microscopy of M hyopneumoniae treated with polycationic ferritin revealed an intermediate, compact, unlabeled layer between the cytoplasmic membrane and an external, heavily labeled layer. Electron microscopy of ferritin-labeled M hyopneumoniae after treatment with trypsin or periodate revealed the intermediate layer to be composed of a trypsin-sensitive protein(s). The outer layer was made of periodate-sensitive carbohydrate(s). Therefore, it appears that proteins in the intermediate layer confer at least part of the total hydrophobicity of the mycoplasmal cell and may contribute to adherence of M hyopneumoniae to target respiratory cells by hydrophobic interactions.

Effect of age on susceptibility of pigs to Mycoplasma hyopneumoniae pneumonia.

The effect of age on susceptibility of pigs to Mycoplasma hyopneumoniae was compared in 2 experiments. In experiment A, pigs 3, 6, and 12 weeks of age were contact-exposed to pigs with established M hyopneumoniae infection for 27 days and then evaluated for pneumonia at necropsy done at 42 days after exposure began. In experiment B, pigs 3 and 11 to 12 weeks of age were contact-exposed for 20 days and then placed in separate, isolated hog houses until necropsy was done at 49 days after exposure began. Evaluations at necropsies revealed that no differences existed between age groups concerning occurrence or severity of pneumonia. Likewise, immunofluorescence and culture evaluations of lung tissue revealed that no significant differences existed among age groups concerning detection of M hyopneumoniae or time of seroconversion to the organism. Under conditions of this study, no differences were detected in susceptibility of pigs between 3 and 12 weeks of age.

Comparison of Mycoplasma hyopneumoniae strains by serologic methods.

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.

Effects of Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine.

Alveolar macrophages were collected at necropsy from pigs inoculated with Mycoplasma hyopneumoniae or Actinobacillus pleuropneumoniae or both and were tested for phagocytic capabilities, using in vitro techniques. Macrophages from noninoculated littermates were used as controls. Alveolar macrophages from pigs inoculated with either M hyopneumoniae or A pleuropneumoniae had significantly (P less than 0.05 to P less than 0.0025) higher phagocytic capacity than that of noninoculated controls. Macrophages from A pleuropneumoniae-inoculated pigs were comparatively more stimulated than were those from M hyopneumoniae-inoculated pigs. Pigs inoculated with M hyopneumoniae and then challenge-exposed with A pleuropneumoniae 2 and 4 weeks later had greatly reduced phagocytosis. Infection with M hyopneumoniae or A pleuropneumoniae caused stimulation of alveolar macrophage functions, and M hyopneumoniae infections may have suppressed phagocytic responses when pigs were challenge-exposed with a secondary pathogen (A pleuropneumoniae). This potential suppression may represent a prediposition of the host by M hyopneumoniae to secondary bacterial infections.

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.

Immunogenicity of experimental Streptococcus equisimilis vaccines in swine.

The protective and complement-fixing antibody immune responses to Streptococcus equisimilis vaccines were evaluated in young, surgically derived, colostrum-deprived swine. Comparable levels of protection against live S equisimilis challenge exposure developed in response to sonic-extract, acid-extract, and whole-cell vaccines combined with incomplete Freund adjuvant. The extract vaccines induced higher levels of complement-fixing antibody than did killed, whole cells. Protection, as well as complement-fixing antibody immune responses, were comparable when swine were given doses of vaccine beginning at 3 or 8 weeks of age.

Modified metabolic-inhibition test for detection of antibodies to Mycoplasma hyosynoviae in swine serum.

A modification of the metabolic inhibition (MI) test was used for detection of antibodies against Mycoplasma hyosynoviae in convalescent swine serum. Supplementation of the MI system with 1% unheated normal rabbit serum, as well as 6% unheated normal guinea pig serum was required for detection of MI antibodies in early phase, postinfection serum and markedly improved detection of MI antibodies in late phase, postinfection serum. Factor(s) supplies by rabbit serum was not supplied by unheated normal swine serum.

Suppressive effect of nonviable Mycoplasma hyopneumoniae on phytohemagglutinin-induced transformation of swine lymphocytes.

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.

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.