Biomedical subjects

M Kobisch

Publications and source records attributed to M Kobisch.

50 records · Page 3Linked to original sources

PubMed1992

Immunological and pathological reactions in piglets experimentally infected with Mycoplasma hyopneumoniae and/or Mycoplasma flocculare.

The aim of this work was to examine in vivo whether infection with Mycoplasma hyopneumoniae (M. hyop) and/or Mycoplasma flocculare (M. floc) would interact and influence the severity of enzootic pneumonia in piglets. Specific pathogen-free, hysterectomy-derived piglets were allocated to six groups and experimentally inoculated with M. hyop. and/or M. floc at the age of 2 or 8 weeks. Clinical symptoms, frequency of coughing and temperature measurement were noted daily. Lung lesions were recorded by post-mortem examination and histological observations. The cross-inoculation with both mycoplasmas did not influence the clinical or the pathological picture of the disease. Evolution of specific and crossreacting antibodies was analyzed by ELISA and immunoblotting. Animals inoculated with M. floc did not develop any lesions but showed a weak antibody response 6-8 weeks post-infection (p.i.). No cross-reacting antibodies against M. hyop proteins were detected. In animals inoculated with M. hyop, the first antibody response was detectable 4-5 weeks p.i. and was stronger in piglets infected at the age of 2 weeks than at the age of 8 weeks. Three cross-reacting antibodies against M. floc proteins with molecular weights of 110, 47 and 33 kDa were detected by antibodies to M. hyop. Experimental infections with both mycoplasmas did not show differences in the pattern of species-specific proteins.

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PubMed1992

Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae.

Seven hysterectomy derived piglets were repeatedly challenged with Mycoplasma hyopneumoniae during the first week of life. Samples of trachea, bronchi and lung tissue collected 2-11 weeks post-inoculation (p.i.) were examined using light and electron microscopy. Autoradiography was used to study in more detail the site of M. hyopneumoniae multiplication. Gross lesions were observed in lung tissue and were characterized by hyperplasia of the epithelium and an increased mononuclear cell accumulation in perivascular and peribronchiolar areas. Mild lesions of the trachea and the bronchi, including epithelial hyperplasia and infiltration of the lamina propria by inflammatory cells, were noted. Electron microscopy showed that, 2-6 weeks p.i., changes in the mid-trachea and bronchi surface consisted of the loss of cilia. Mycoplasmas covered tufts of cilia remaining on the epithelial cell surface. Scanning and transmission electron micrographs showed that they were predominantly found closely associated with the top of cilia. No specialized terminal structure could be seen and no mycoplasma cells were identified lying free in the lumen nor in close contact with the plasma membrane of cells or microvilli. Some fine fibrils radiating from one mycoplasma to another or to cilia were seen at higher magnification by scanning electron microscopy. Six to eleven weeks p.i., a disrupted epithelial surface lacking cilia was observed. Cells were desquamated and shed into the lumen with cellular remains containing droplets of mucus. Autoradiography revealed that label corresponded to the observed mycoplasma distribution. At the top of cilia, a high density of labeling was visible in the zone of high mycoplasma concentration. Therefore, incorporation of the label in the mycoplasma is proof or their multiplication in the trachea. The intimate association between the mycoplasma and cilia may be an important factor in the pathogenesis of the disease caused by M. hyopneumoniae (swine enzootic pneumonia).

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PubMed1992

A specific DNA probe for detecting Mycoplasma hyopneumoniae in experimentally infected piglets.

Mycoplasma hyopneumoniae is the primary agent of swine enzootic pneumonia. Because of fastidious growth requirements and its serological cross-reactions with other porcine mycoplasmas, we developed a specific DNA probe for its detection. A partial genomic library of M. hyopneumoniae was constructed in plasmid pBR 322 using Hind III chromosomal fragments. The recombinant plasmids were screened by differential hybridization with M. flocculare and M. hyorhinis genomic DNA probes. One non-hybridizing recombinant plasmid was selected and its 1.65 kbp insert (designated I141) tested for specificity against genomic DNA from numerous mycoplasmas, other bacteria species and DNA from lung tissue of specific pathogen free (SPF) piglets. The 32P labelled I141 could detect specifically down to 400 pg of M. hyopneumoniae genomic DNA. To test the suitability of the I141 probe for the laboratory diagnosis of M. hyopneumoniae infections, we used clinical tracheobronchial specimens from piglets which were experimentally infected with M. hyopneumoniae. The results with hybridization on each specimen were compared to findings with an immunofluorescence test. Of the clinical specimen tested, there was agreement in the two tests of 63%.

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PubMed1992

In vitro colonization of porcine trachea by Mycoplasma hyopneumoniae.

Porcine tracheae maintained in culture were used in order to study the colonization by Mycoplasma hyopneumoniae. Rings excised from tracheae of newborn piglets were infected with M hyopneumoniae strain BQ 14 and, after different incubation times, were examined by light and electron microscopy. Non-infected tracheal mucosae maintained a normal appearance for several days. Infected tracheal rings showed progressive colonization with concomitant progressive damage to the mucosal surface. Early on during the infection, few mycoplasmas occurred over a ciliated epithelium. As the infection progressed, there was gradual loss of cilia; mycoplasmas tended to form microcolonies and to accumulate over the remaining ciliated cells. Mycoplasmas, first seen at the apex of the cilia, were then seen deeper in the inter-ciliary space; some were even seen in contact with microvilli. In histological investigation, the final stage of the infection was characterized by a marked destruction of the epithelium with exfoliation of the epithelial cells. Infected mucosae showed typical damage caused by M hyopneumoniae, namely reduction of ciliary activity after 5 days, loss of cilia, and sloughing of ciliated cells. Our data indicate that porcine tracheal organ culture can be advantageously used to study colonization by M hyopneumoniae.

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PubMed1991

Cloning and expression of a species-specific early immunogenic 36-kilodalton protein of Mycoplasma hyopneumoniae in Escherichia coli.

Mycoplasma hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, synthesizes a 36-kDa protein which is an early and strong immunogenic factor in experimentally and naturally infected swine. The gene encoding this protein was cloned by screening a gene library of M. hyopneumoniae DNA with rabbit hyperimmune serum made against whole M. hyopneumoniae cells and convalescent-phase swine serum. Analysis of the recombinant protein expressed in Escherichia coli by immunoblot techniques showed that the protein is expressed in E. coli in its full length and does not cross-react with proteins from M. flocculare or M. hyorhinis. Genetic analysis showed that the gene was expressed from the lac promoter of the vector and seems to be translationally initiated from its own ribosome binding site. Subcloning in a transcriptional fusion vector to optimize expression resulted in production of the 36-kDa protein in E. coli at levels up to 30% of total protein.

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PubMed1991

Adherence of Pasteurella multocida isolated from pigs and relationship with capsular type and dermonecrotic toxin production.

Pasteurella multocida can often be isolated from pneumonic lungs in pigs. There is little information about the pathogenesis of this infection. Attachment of microorganisms to eucaryotic cells is considered to be a prerequisite for colonization of the host in the pathogenesis of bacterial infections. Forty-seven P multocida strains isolated from pigs in France, and belonging to capsular type A or D were tested for their ability to agglutinate human erythrocytes, and to adhere to tracheal and lung cells. Each isolate was tested for dermonecrotic toxin production. Adherent strains were further observed by electron microscopy to look for attachment structure. Only type A strains agglutinated human O erythrocytes, but no relationship was observed between hemagglutination and dermonecrotic toxin production. The results of the adherence tests showed a greater affinity (P less than 0.05) of type A strains for lung cells (50% were adherent, whereas only 20% of type D strains were adherent) but did not reveal any correlation between adherence and the presence of dermonecrotic toxin. Microscope observations showed that these P multocida strains did not possess any pili-like structures. In conclusion, by means of the adherence test we were able to demonstrate a stronger adherence of type A strains and this adherence did not seem to be related to pili-like structures.

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PubMed1990

Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets.

Maternal antibody to an outer membrane 68-kilodalton (kDa) protein of Bordetella bronchiseptica was shown to be protective in experiments on specific-pathogen-free piglets. After challenge with B. bronchiseptica, 100% (n = 19) control piglets from nonimmunized sows developed pneumonia, coughing, and sneezing, and 74% of the animals developed severe atrophic rhinitis. In 12 piglets from a sow immunized with 68-kDa protein, pneumonia occurred only in 34% of offspring, coughing was reduced, the duration of coughing bouts was shortened, and severe atrophic rhinitis occurred in one animal only (8%). The difference in the occurrence of atrophic rhinitis and of pneumonia in immunized and nonimmunized offspring was statistically significant (P less than 0.05). Sera of protected piglets had high titers (enzyme-linked immunosorbent assay) of antibodies that showed a high specificity for the 68-kDa protein isolated from B. bronchiseptica, whereas their reactivity with an analogous 69-kDa protein isolated from Bordetella pertussis was low or absent. The 68-kDa protein of B. bronchiseptica appeared to be the major protective antigen in B. bronchiseptica infection; however, isolated protein alone did not induce such a solid protection, as observed in a previous study after the application of an effective whole cell vaccine.

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PubMed1989

An evaluation in pigs of Nobi-Vac AR and an experimental atrophic rhinitis vaccine containing P multocida DNT-toxoid and B bronchiseptica.

The trial involved eight large white sows obtained from a closed experimental specific pathogen free herd. Four sows (two each for an experimental vaccine and for Nobi-Vac AR) were vaccinated twice (eight weeks and two weeks before parturition) with 2 ml of vaccine administered intramuscularly. Two unvaccinated sows were used as an infected control group and two unvaccinated sows served as an uninfected control group. Forty-six piglets (28 from vaccinated sows and 18 from unvaccinated sows) were challenged by intranasal instillation of Bordetella bronchiseptica at two days of age and Pasteurella multocida type D, dermonecrotic toxin at seven days of age. Among the infected control group some piglets died and there were clinical signs of pneumonia and severe turbinate atrophy. In the vaccinated groups the results showed that immunisation of the pregnant sows had provided a good level of antibodies, which were transmitted to their offspring. There was a significant reduction in the clinical signs and no lesions were observed in the group vaccinated with the experimental vaccine and only moderate atrophy of the turbinates in the Nobi-Vac AR group. B bronchiseptica and P multocida were never recovered from the lungs of the vaccinated groups and in the nasal cavities their frequency declined with age.

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PubMed1987

Comparison of enzyme-linked immunosorbent assay (ELISA) and indirect hemagglutination (IHA) in experimental Mycoplasma hyopneumoniae infection of pigs.

Twenty-five hysterectomy-derived piglets were infected intratracheally at 16 days of age with a field strain of Mycoplasma hyopneumoniae. Twenty uninfected piglets constituted the control group. Clinical symptoms were noted daily and sera collected weekly. The 45 pigs were killed from 1 to 20 weeks after infection. M. hyopneumoniae induced a temperature response, coughing and pneumonia. After 10 weeks, a noticeable regression of the macroscopic lung lesions was observed. The infection was followed by an antibody response, and the antibody titers reflected the disease stages. ELISA appears to be the most sensitive procedure to detect early and late antibody production.

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PubMed1985

[Respiratory diseases of swine: some epidemiologic aspects].

Epidemiological surveys enable to know better the aetiology of respiratory diseases. The surveys carried out in slaughter-houses in Brittany (France) in 1980 and 1981, show a worrying situation concerning porcine respiratory diseases. Microbiological studies brought out the preponderance of the isolation of Mycoplasma hyopneumoniae, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis and Actinobacillus suis. Some patterns of experimental infection with Mycoplasma hyopneumoniae and Bordetella bronchiseptica are exposed.

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PubMed1985

Stimulation of immunoglobulin-containing cells and isotype-specific antibody response in experimental Mycoplasma hyopneumoniae infection in specific-pathogen-free pigs.

The development of immunoglobulin-containing cells and antigen-specific plasma cells in retropharyngeal and bronchial lymph nodes, in lung tissue, and in nasal mucous membrane was followed during an experimental infection of pigs with Mycoplasma hyopneumoniae. An enzyme-linked immunosorbent assay was used to follow stimulation of the immunoglobulin isotypes (classes) M, G, and A directed against the infectious agent in tracheobronchial secretions and systemically. A significant increase of antigen-specific plasma cells in lymph nodes and lung tissue as well as antibodies in tracheobronchial secretions was demonstrated 2 weeks after infection. This specific reaction was followed by a marked increase of immunoglobulin-containing cells in lymph nodes and lung tissue. The data are discussed in terms of autoimmune reactions in the pathogenesis of enzootic pneumonia.

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PubMed1985

Evaluation of Bordetella bronchiseptica vaccines in specific-pathogen-free piglets with bacterial cell surface antigens in enzyme-linked immunosorbent assay.

The progenies of specific-pathogen-free sows which had been immunized with Bordetella bronchiseptica vaccines of various origin before parturition were challenged intranasally with B. bronchiseptica within 5 days of birth. Sera of piglets were taken weekly and investigated by enzyme-linked immunosorbent assay against a mixture of B. bronchiseptica cell surface antigens containing curled fibers and fimbriae, lipopolysaccharide, and a mixture of proteins mostly derived from the outer membrane. The serological response to this antigenic mixture was paradoxical; the highest titers were obtained with the least effective vaccines. Antibodies which did relate to protection were oriented against the outer-membrane-derived proteins, one of which, of 68,000 molecular weight, appeared to be particularly important for two reasons. First, its concentration within the antigenic mixture was dependent upon cultural conditions; of all the proteins present in virulent strains, it was the first to disappear upon modulation. Second, it was absent from a strain which was unable to induce atrophic rhinitis in specific-pathogen-free piglets. Although all vaccines tested had some beneficial effect on the various clinical manifestations of the disease, only two vaccines were effective (P less than 0.001) in the prevention of nasal pathological changes. These two vaccines also stimulated the highest titers against the 68,000-molecular-weight protein. A mouse protection test utilizing a lethal intraperitoneal challenge failed to monitor the efficacy of vaccines for protection against atrophic rhinitis.

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PubMedDate not supplied

The Mycoplasma hyopneumoniae plasma membrane as a vaccine against porcine enzootic pneumonia.

From an economical standpoint, the colonization of the respiratory epithelium by Mycoplasma hyopneumoniae, the mollicute responsible for enzootic pneumonia in pigs which leads to a reduction in the weight gain/feed ratio, has serious consequences in very young animals; we show here that its is possible to efficiently protect these animals with the colostral antibodies raised in sows immunized during their gestation with M. hyopneumoniae BQ14 membranes. In an experiments using 9 sows and 75 piglets, the vaccinal preparation was found to protect 70 to 100% of the piglets (depending upon the presentation of the immunogen) as well as being totally innocuous. The best results were obtained with an adjuvant made of hydroxide aluminium gel. Of the piglets born to non-immunized sows, 8.3% exhibited typical pneumonic lesions. The vaccination procedure was found to enhance the elimination of M. hyopneumoniae from the respiratory tract of the piglets, as in 95% of the protected animals, the microorganisms could no longer be isolated. Conversely, M. hyopneumoniae was isolated from 89% of the piglets born to unvaccinated sows. A correlation was found between the protective effect and the rise in the titre of circulating antibodies directed against M. hyopneumoniae surface antigens. These results show that the plasma membrane of M. hyopneumoniae is a potential vaccine against porcine enzootic pneumonia. In particular, passively transmitted immunity via the colostrum provides the most precocious means of protection for the animals.

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PubMedDate not supplied

Production of muraminidase-released protein (MRP), extracellular factor (EF) and suilysin by field isolates of Streptococcus suis capsular types 2, 1/2, 9, 7 and 3 isolated from swine in France.

A total of 323 isolates of Streptococcus suis recovered from diseased or healthy pigs in France were serotyped. The presence of virulence-related proteins, Muraminidase-Released Protein (MRP), Extracellular Factor (EF) and Suilysin was also studied in 122 isolates of capsular types 2, 1/2, 9, 7 and 3 to evaluate their implication in virulence of S. suis. Capsular types 2, 1/2, 9, 7 and 3 were the most frequently detected (93%), with 69% for the capsular type 2 alone. Capsular types 2, 1/2, 9, 7, 3, 1, 4, 8, 18, 10 and 12 were isolated from diseased pigs, whereas types 2, 7, 9, 1/2, and 3 originated from the nasal cavities or tonsils of healthy animals. Most of the S. suis type 2 isolates recovered from diseased pigs carried MRP+ EF- Suilysin- (46%) or MRP+ EF+ Suilysin+ (28%) phenotypes. The MRP+ EF- Suilysin- phenotype was also detected in 67% of S. suis type 2 strains isolated from healthy pigs. The production of the virulence-related proteins was less frequently found in S. suis types 1/2, 9, 7 and 3 recovered either from diseased or healthy pigs. In this study, all the capsular type 1/2 strains were MRP+ EF- Suilysin- and all the S. suis type 7 harboured an MRP- EF- Suilysin- phenotype. The MRP- EF- Suilysin- phenotype was found in S. suis types 2, 3, 7 and 9 isolated from septicaemia, meningitis, pneumonia, and pleurisy. These results suggest that the presence of these proteins should not be used as a single condition for classifying the virulence of a field isolate in France.

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