Hydrogen peroxide production and myo‐inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo‐inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo‐inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo‐inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence.     

SDS-PAGE separation of dermatophyte antigens,and western immunoblotting in feline dermatophytosis

Water-soluble antigens liberated from the disrupted mycelium of nine dermatophytes (seven isolates ofMicrosporum canis, one each ofMicrosporum gypseum andTrichophyton mentagrophytes) were compared by analytical slab SDS-PAGE. No substantial differences were observed between the protein bands of theM. Canis isolates, but certain distinctive bands were apparent in the other two species examined. Western immunoblotting usingM. canis-derived antigens separated by SDS-PAGE was used to investigate the humoral immune response in 79 cats with naturally-occurring dermatophytosis (72 withM. canis, six withM. gypseum and one withT. mentagrophytes) and this information was compared to results of immunoblots from 46 control (non-dermatophyte exposed) cats. Seven dominant bands (bands which occurred frequently and stained heavily) were identified in immunoblots from the dermatophyte-infected cats with apparent molecular weights varying between 39 and 120 kD. None of these bands were totally specific markers for dermatophytosis as a variable proportion of the control cats showed reactivity to all these proteins. However, most (73%) of the dermatophyte-infected cats showed reactivity to six or seven of the identified bands whereas most (80%) of the control cats showed reactivity to between zero and three of these bands (p<0.005). Western immunoblotting could be used to select individual immunodominant antigens for further evaluation of protective (cell-mediated) immunity.     

Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis

Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium; preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis.     

Effect of climatic conditions on enzootic pneumonia of pigs

Enzootic pneumonia of pigs is a common worldwide problem affecting mainly growing pigs. It is caused byMycoplasma suipneumoniae (M. hyopneumoniae) but the pneumonia is usually complicated byM. hyorhinis and bacteria. The experimental evidence on the effect of temperature, UV light and drying on the survival ofM. suipneumoniae is reviewed and related to the data available onM. pneumoniae M. mycoides subsp.mycoides andM. gallisepticum which cause respiratory disease in man, cattle and chickens respectively. The external and internal climatic conditions which influence the severity of enzootic pneumonia in housed pigs are discussed. Possible further experiments withM. suipneumoniae are discussed in relation to the problem of cultivating one of the most fastidious of all known mycoplasmas.Presented at the Seventh International Biometeorological Congress, 18–25 August 1975, College Park, Maryland, USA.     

Factors influencing microbiological assay of cell-culture mycoplasmas

Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas. These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and N01-GM-6-2119 from the National Institute of General Medical Sciences.     

Immunohistochemical staining of macrophages in the skin lesions of leprosy: the role of antibody to mycobacteria in human serum and various polyclonal immune rabbit antisera

Summary Immunohistochemical staining of tuberculoid and lepromatous leprosy skin lesions was performed using various rabbit antisera. Macrophages in both stained with serum containing antibodies against lysozyme and alpha-1-antitrypsin, while macrophages in lepromatous leprosy also reacted with other antibodies. An immunoglobulin fraction of positive serum stained following pepsin digestion, indicating that reactivity was not Fc dependent. Positive serum contained antibody againstMycobacterium butyricum, which caused macrophage staining, since affinity-purified antibody did not stain and absorption withM. butyricum removed staining. Staining was also produced by serum of subjects with leprosy or a positive tuberculin test. By immunoblotting, the anti-mycobacterial antibody was directed against surface components ofM. butyricum of molecular weights 20 000–70 000. Electron microscopy showedM. leprae in phagolysosomes of macrophages, while immunoelectron microscopy demonstrated labelling along bacterial cell membranes. Therefore, macrophages in lepromatous leprosy skin lesions stain because they containM. leprae, which reacts with antibody to eitherM. leprae, M. tuberculosis or atypical mycobacteria in human serum and with antibody toM. butyricum in serum from rabbits immunized with various antigens and Freund's complete adjuvant. These results indicate that immunohistochemical studies on leprosy are misleading if performed using intact polyclonal immune sera rather than affinity purified or monoclonal antibodies.     

Mink lung cells as a tool for detection ofMycoplasma hyorhinis contamination in cell cultures and virus stocks

Summary In an extensive host range study ofM. hyorhinis mink lung cells (MvlLu, ATCC CCL 64) were found to be the cells of choice for the propagation of this mycoplasm, which otherwise is often difficult to grow in a cell-free medium. Furthermore, rapid plaque assay and plaque purification procedures were developed forM. hyorhinis. The titer ofM. hyorhinis grew to 1×10⁷ to 1×10⁸ pfu/ml within three d postinoculation on mink lung cells. DNA restriction enzyme analysis of the genome ofM. hyorhinis was performed. Endonucleases Bst EII and Xho I are the most suitable enzymes for cleavingM. hyorhinis DNA into distinct fragment patterns. Thus, the use of the combination mink lung cells for mycoplasma growth with subsequent restriction enzyme analysis leads to an unamibiguous detection and identification toM. hyorhinis strains even in minute amounts.     

Serologic characterization of aMalacosoma disstria Hübner (Lepidoptera: Lasiocampidae) cell line

Summary The identity of a cell line derived from hemocytes ofMalacosoma disstria was investigated serologically by using complement fixation, double diffusion, immunoelectrophoresis in agarose, and acrylamide gel electrophoresis. TheM. disstria cell line antiserum gave a specific cross-reaction with its homologous antigen and withM. disstria larval antigen, although cross-reactions with cell lines fromAedes aegypti, Bombyx mori, andChoristoneura fumiferana and with larval antigens ofB. mori andC. fumiferana were also produced. Antisera againstA. aegypti andB. mori cell lines showed very similar cross-reaction with both the cell line antigens. Also, these two antisera gave a strong reaction withB. mori larval antigen, but no reaction withA. aegypti larval antigen. These tests confirm the identity of ourM. disstria cell line. Also, they indicate: (a) that theA. aegypti andB. mori cell lines tested are similar, and (b) that they are closely related toB. mori, but not at all related toA. aegypti. Contribution No. 233, based on a paper presented at the 23rd Annual Meeting of the Tissue Culture Association, Los Angeles, June 5–8, 1972.     

Acquired resistance to ixodid ticks induced by tick cement antigen

Antisera from guinea pigs made resistant to infestation with an ixodid tick of east and central Africa,Rhipicephalus appendiculatus, were used to identify the tick antigens they recognized by immunoblotting. Most of the antigens were found in tick salivary glands and in tick attachment cement. Antisera fromR. appendiculatus-resistant guinea pigs also recognized some salivarygland antigens in ticks of other species (R. pulchellus, R. evertsi, Amblyomma variegatum andA. gemma). Antibodies against the most strongly recognizedR. appendiculatus antigen, a 20-kDa molecule, were only poorly reactive with similar-sized molecules in the other ticks. A 94-kDa antigen, which appeared to have broader cross-reactivity, was purified fromR. appendiculatus attachment cement, and a monospecific rabbit serum was raised against it. This antiserum clearly recognized a molecule of similar molecular weight inR. pulchellus andR. evertsi. Intravenous inoculation of rabbits with the purified molecule elicited delayed-type hypersensitivity to the antigen. The hypersensitive rabbits demonstrated resistance to feeding ofR. appendiculatus ticks but slight enhanced feeding ofR. pulchellus ticks. These results are discussed with respect to their relevance for artificial induction of tick-feeding resistance.     

Proteolytic processing of the cilium adhesin MHJ_0194 (P123J) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules

Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non‐ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C‐terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.     

Preparation of h and m antigens ofHistoplasma capsulatum free of heterologous antigens

Salt gradient elution of crude histoplasmin on CM-Sepharose CL6B at pH 3.0 was used in essentially a one-step procedure to isolate the h, m, and non-m antigens ofHistoplasma capsulatum and free them of any c antigen common to other pathogenic fungi. The h antigen fraction was pure; the isolated m and non-m antigens contained less than 0.1% of the c antigen present in the original preparation. The residual c antigen in these fractions was removed by affinity chromatography on Affigel-10 coupled to aBlastomyces dermatitidis fungal antiserum. The final preparations of h, m, and non-m were pure when tested by immunodiffusion (ID), sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), or electrofocusing. The antigens were serologically stable at neutral pH for at least three months at 10°C. The relative molecular weights, isoelectric points, and amino acid composition of the antigens are described.     

Serological difference betweenErwinia herbicola strains of plant and human origin

FiftyErwinia herbicola isolates obtained from host plants were examined in an agglutination reaction with antiserum prepared againstE. ananas (E. herbicola) strain CCM 2407 antigen of plant origin and with antiserum prepared againstEnterobacter agglomerans strain CNCTC M 269 antigen of human origin. In tests with strain CCM 2407 antiserum, 56% isolates showed a positive reaction, while in tests with strain CNCTC M 269 antiserum only 14 % isolates showed a positive reaction. AmongE. herbicola isolates which showed a positive reaction with CCM 2407 antiserum 18 % showed a positive reaction with the CNCTC M 269 antiserum too. Our results confirmed the serological heterogeneity ofE. herbicola population. In spite of the difference in the origin of the two antigens used for the preparation of antisera (plant, human; Japan, Czech Republic) our results indicate that some of ourE. herbicola strains andE. agglomerans strain CNCTC M 269 are serologically identical.     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody

Summary Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation. This allows the same field on a slide to be viewed for presumptive diagnosis of any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis ofM. hyorhinis strains using fluoresceinated antibody. The use of this method plus a cultural procedure will permit identification of the “noncultivable”M. hyorhinis strain DBS 1050.     

猪肺炎支原体Mhp366-N蛋白多克隆抗体的制备及应用

【背景】猪肺炎支原体是猪的一种重要的病原。该菌的研究工具较少,特别是缺少开展其致病机制研究需要的抗体。【目的】制备猪肺炎支原体Mhp366-N蛋白抗体并确定其应用范围和使用时的最佳稀释倍数。【方法】Escherichia coli BL21(DE3)-pET28a(+)-mhp366-N重组菌诱导表达Mhp366-N蛋白并纯化。纯化的蛋白免疫小鼠制备多克隆抗体。用免疫印迹和免疫荧光方法检测猪肺炎支原体AH株感染3D4/21细胞后的Mhp366蛋白,确定2种方法中Mhp366-N多克隆抗体的最佳稀释倍数;之后检测临床采集的猪肺泡巨噬细胞中的猪肺炎支原体;最后以免疫组化试验检测猪肺炎支原体感染的肺细胞。【结果】纯化的Mhp366-N蛋白纯度超过85%,免疫小鼠制备的抗血清效价在1:128 000-1:512 000之间。在免疫印迹试验中Mhp366-N多克隆抗体的最佳工作浓度为1:100 000稀释,免疫荧光试验中Mhp366-N多克隆抗体的工作浓度范围在1:1 000-1:10 000 000,其可用于临床采集的猪肺泡巨噬细胞和细胞系中猪肺炎支原体的检测。免疫组化试验结果显示猪肺炎支原体能够进入猪肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞。【结论】制备的Mhp366-N多克隆抗体为猪肺炎支原体致病机制研究提供了良好的研究工具。     

Transcription Analysis of the Porcine Alveolar Macrophage Response to Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod)-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.     

Serum requirements necessary for the opsonophagocytosis ofescherichia coli O73:K92:H1

TheEscherichia coli O73:K92:H1 serotype, which possesses a capsular antigen immunochemically similar to the capsule of the group C meningococcus, is demonstrated in this study to be resistant to phagocytosis by normal human PMNs and serum and to be dependent upon immune antibody and presumably the classical complement pathway for opsonization. Using both a luminol-dependent chemiluminescence assay and an in vitro bactericidal system, we examined, in both the absence and presence of complement, the opsonic activity of IgM ang IgG antisera. Of the various antisera tested, only those sera cross-reactive with the K92 capsular antigen were found to be opsonic both in vivo and in vitro, while somatic O or lipid A antisera demonstrated no activity. In in vitro studies with capsular IgM and IgG antisera, only IgG demonstrated opsonic activity without complement, whereas IgM required complement for opsonization of O73:K92:H1. These data demonstrate that antisera directed toward capsular antigens are opsonic for this phagocytosis-resistantE. coli, and that complement is a necessity for opsonization in the absence of sufficient capsular IgG antibodies.     

Antibodies to selected pathogens in wild boar (Sus scrofa) from Catalonia (NE Spain)

From 2004 to 2007, blood samples from 273 healthy wild boars (Sus scrofa), culled during the hunting season, were obtained in three areas of Catalonia (NE Spain): Pyrenees, Sant Llorenç del Munt i l’Obac Natural Park (SLM), and Ports de Tortosa i Beseit National Hunting Reserve (PTB). We investigated the presence of antibodies against classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine vesicular disease virus (PVDV), porcine respiratory and reproductive syndrome virus (PRRSV), Aujeszky’s disease virus (ADV), porcine influenza A virus (PIV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Salmonella spp., and Toxoplasma gondii. Four wild boars were suspicious for CSFV, but the infection was discarded with a virus neutralization test, and infection with a border disease virus was confirmed. Negative results were obtained against ASFV and PVDV. Antibodies were detected against PRRSV (3%), ADV (0.8%), PIV (6.4%), PCV2 (64.6%), PPV (54.7%), M. hyopneumoniae (26.6%), E. rhusiopathiae (5.3%), Salmonella spp. (11.3%), and T. gondii (43.5%). In SLM, we detected a higher seroprevalence for PIV and M. hyopneumoniae and a lower seroprevalence for E. rhusiopathiae than in the other two areas. In PTB, seroprevalence was higher for PPV, Salmonella spp., and PCV2. Adult wild boar displayed higher seroprevalence for PPV, PIV, and M. hyopneumoniae, whereas presence of antibodies for Salmonella spp. was higher in juveniles compared with adults and piglets.

     

Immunoelectrophoretic characteristics ofOphiobolus graminis Sacc. as an aid in classification and determination

Antigens from four cultures ofO. graminis were compared immunoelectro-phoretically. Each culture produced a characteristic immunogram. More common antigens were found between the two cultures isolated from wheat or the two cultures isolated from oat than between a wheat and an oat isolate. Cell-wall antigens were the best reference antigens for serologic analysis of strain relationship. O. graminis antisera were cross-reacted with antigens from a number of other species of fungi. Relatively few of these cross-reacted with antisera to cell-wall antigens whereas more cross-reacted with antisera to whole-cell antigens.Immunoelectrophoretic analysis of antigens from a range of isolates ofO. graminis indicates specific immunograms which can be determined and separated from the immunograms developed by all other fungi when tested againstO. graminis antiserum. Immunoelectrophoresis can therefore be used as an aid in determiningO. graminis.