Publications (39)11.8 Total impact

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    H Pfützner · K Sachse ·
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    ABSTRACT: Bovine diseases due to Mycoplasma bovis can cause considerable economic losses in cattle production. While the pathogen is principally responsible for therapy-resistant mastitis on large dairy farms, on smaller farms the typical mycoplasma diseases are calf pneumonia and arthritis. Moreover, the pathogen is able to cause genital disorders. M. bovis infection can be controlled effectively only if appropriate measures are implemented at the earliest possible stage. Since immunoprophylaxis and antibiotic treatment are known to be ineffective, control measures must include the introduction of strict hygiene standards, the restriction of animal movement out of infected herds and the culling of clinically diseased animals and shedders of the mycoplasma (the latter only in the case of mastitis and genital disorders). In this review, symptoms of the various diseases caused by M. bovis are described and characteristics of the course of infection are outlined. To clarify the origin and spread of the infection, the authors describe the main properties and reservoirs of the pathogen and summarise experimental evidence on modes of transmission to susceptible organs. As effective diagnosis is a prerequisite for the introduction of early control measures, the advantages and disadvantages of currently used diagnostic methods are discussed in detail. It is a serious shortcoming if testing for mycoplasmas is not included in routine bacterial examination of clinical samples. As a consequence, some M. bovis infections will remain undetected and outbreaks cannot be controlled properly. Finally, practical recommendations are given for prevention and control, including the formation of mycoplasma-free herds.
    Revue scientifique et technique (International Office of Epizootics) 01/1997; 15(4):1477-94. · 0.91 Impact Factor
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    ABSTRACT: Mycoplasma (M.) bovis cytadhesion was studied using permanent embryonic bovine lung (EBL) cells as host system. Adherence rates were found to be strongly dependent on temperature and the mycoplasma-to-EBL ratio near the point of saturation of the attachment isotherm was determined to be 225 : 1. Mild trypsinization of viable M. bovis cells caused a measurable decrease of adherence indicating that surface proteins, among them the P26 antigen, played a major part as adhesion factors. Neuraminidase treatment of mycoplasmas led to a drastic reduction of adherence rates, which emphasizes the importance of sialyl moieties in adhesive interactions. The ability of the P26 antigen, a hydrophilic 32-kDa protein, to function as an adhesin was confirmed using a competitive adherence assay, in which the HPLC-purified protein was shown to reduce mycoplasma adhesion. These data complement previous findings obtained with the corresponding monoclonal antibody (MAb) 4F6. In further inhibition experiments, it could be demonstrated that MAb 1E5, which is directed against a common epitope of at least three members of the Vsp (variable surface protein) family of M. bovis, was also capable of decreasing mycoplasma attachment to EBL cells. This is the first evidence of possible involvement of Vsps in cytadhesion. In an effort to identify more putative adhesion proteins of this organism, the reverse adherence screening assay was used, a procedure based on the specific binding of labelled mammalian tissue culture cells to Western-blotted mycoplasmal proteins.
    Zentralblatt für Bakteriologie: international journal of medical microbiology 07/1996; 284(1):80-92. DOI:10.1016/S0934-8840(96)80157-5
  • H Hotzel · K Sachse · H Pfützner ·
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    ABSTRACT: Two methods are suggested that allow direct detection of Mycoplasma bovis from clinical samples, i.e. milk and nasal swabs, respectively. Milk samples were trypsinized in the presence of Triton X-100 and passed through a DNA-binding filter membrane, from which DNA was extracted and subjected to PCR. The detection limit was 500 cfu ml-1 on agarose gels and 50 cfu ml-1 after Southern hybridization so that the method can be used to monitor low-titre samples from animals at the subclinical stage. Results became available within 24 h, thus rendering the procedure more rapid than ELISA and culture techniques. Six other methods designed for milk or other complex samples were tested in this study, but found unsatisfactory. Rapid and specific detection of the pathogen by PCR from nasal mucus treated with lysis buffer and proteinase K was demonstrated for swabs taken from experimentally infected calves. Both methods represent useful tools for effective livestock monitoring and single-animal diagnosis.
    The Journal of applied bacteriology 06/1996; 80(5):505-10. DOI:10.1111/j.1365-2672.1996.tb03249.x
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    ABSTRACT: The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important contaminants of cell cultures such as M. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1-2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.
    Cytotechnology 02/1994; 16(2):67-77. DOI:10.1007/BF00754609 · 1.75 Impact Factor
  • Martin Heller · Evelyn Berthold · Horst Pfützner · Rolf Leirer · Konrad Sachse ·
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    ABSTRACT: An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mAb) was developed to detect Mycoplasma (M.) bovis in milk samples from cattle. With this procedure, 1 x 10(5) colony forming units per ml (cfu/ml) milk were routinely detectable. No cross-reactions to other bovine mycoplasma species were observed. Both the sensitivity of 80.6% and the specificity of 94.9% are sufficient for its use in diagnosis of clinical mastitis. The sensitivity could be increased by 10% after introduction of 48-hour pre-incubation of samples. This allowed recognition of cows shedding M. bovis amounts of 10(3) cfu/ml in their milk, which is typical for subclinical cases. Screening of milk samples by means of this antigen capture ELISA has advantages over culture methods in terms of speed and potential to monitor large herds, thereby permitting early culling of infected animals to reduce transmission of the pathogen to non-infected animals.
    Veterinary Microbiology 11/1993; 37(1-2):127-33. DOI:10.1016/0378-1135(93)90187-C · 2.51 Impact Factor
  • K Sachse · H Pfützner · M Heller · I Hänel ·
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    ABSTRACT: The attachment of Mycoplasma bovis to permanent embryonic bovine lung (EBL) cells was studied in order to identify factors participating in the adhesion process. A monoclonal antibody directed against a 26 kDa protein of M. bovis was shown to reduce cytadherence of strains 120 and 454 by 46% and 70%, respectively. In uninhibited assays, strain 120 which exhibits an intense 26 kDa band in electrophoretic protein patterns adhered more strongly to EBL cell monolayers than strain 454 whose corresponding band is considerably weaker. The findings indicate involvement of the 26 kDa protein in M. bovis adherence. In further inhibition experiments, the ability of N-acetyl-neuraminlactose, glycophorin and dextran sulfate to significantly reduce adherence could be demonstrated. This suggests participation of sialic acid residues and probably also sulfatide groups as binding receptors. The data point to a complex adhesion mechanism with similarities to M. pneumoniae.
    Veterinary Microbiology 10/1993; 36(3-4):307-16. DOI:10.1016/0378-1135(93)90097-Q · 2.51 Impact Factor
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    K Sachse · H Pfützner · H Hotzel · B Demuth · M Heller · E Berthold ·
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    ABSTRACT: Mycoplasma bovis, the main causative agent of mycoplasmal mastitis, arthritis and pneumonia in cattle, causes considerable economic losses. Veterinary hygiene measures would be most effective if introduced at an early stage, especially the culling of cows shedding the pathogen for the control of mastitis. It is therefore crucial to ensure that diagnostic methods are available which can perform rapid and specific detection of the agent at acceptable costs. Six different detection methods have been compared and evaluated in terms of performance parameters and suitability for routine diagnosis. Conventional M. bovis isolation and identification from culture is the only technique used for routine diagnosis at present. However, this process is rather laborious and time-consuming, and final results are available only after several days. Enzyme-linked immunosorbent assay (ELISA) techniques can be used to screen for M. bovis antibodies or antigens in clinically-diseased animals. Detection of the agent in subclinical cases was accomplished in pre-incubated samples by an antigen capture ELISA involving a monoclonal antibody. Whole-cell protein patterns generated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to identify and classify field isolates. Nucleic acid hybridizations using probes of defined specificity were conducted both as filter dot blot assay and in solution with ribosomal ribonucleic acid as the target. The latter was found to be potentially suitable for the screening of biological samples, although problems due to high background and reduced specificity remained. Finally, the presence of M. bovis cells in culture supernatant and in milk samples was demonstrated using the polymerase chain reaction. This procedure is potentially superior to all others currently available, due to its high sensitivity, specificity and speed. However, a number of practical problems must be solved prior to full-scale introduction of this technique for routine diagnosis.
    Revue scientifique et technique (International Office of Epizootics) 07/1993; 12(2):571-80. · 0.91 Impact Factor
  • H Hotzel · B Demuth · K Sachse · A Pflitsch · H Pfützner ·
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    ABSTRACT: Detection of Mycoplasma bovis by traditional culture methods is rather time-consuming and is often hampered by bacterial contamination. The development of a rapid and specific alternative is, therefore, an important prerequisite in improving the diagnosis of bovine diseases caused by this agent. The authors have successfully used nucleic acid probes containing genomic restriction fragments of M. bovis cloned into the plasmid vector pUC19 for species-specific detection by dot blot hybridisation of small quantities of M. bovis deoxyribonucleic acid (DNA). The problem of direct M. bovis detection from contaminated milk could not be solved using this procedure. Therefore, further research was conducted using in vitro DNA amplification by polymerase chain reaction (PCR). Species-specific nucleic acid probes were sequenced and suitable PCR primers selected. Using the PCR procedure, ten colony-forming units (CFU) were detected from broth cultures and, after DNA isolation, the equivalent of 1 CFU was detected. Direct detection of M. bovis from biological samples proved extremely difficult due to protein interference. It was shown, however, that direct PCR detection from milk is possible after effective protein removal by combined extraction and protease digestion.
    Revue scientifique et technique (International Office of Epizootics) 07/1993; 12(2):581-91. · 0.91 Impact Factor
  • A Brys · H Pfützner · H Bocklisch · H Weigel ·
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    ABSTRACT: On a cattle farm latently infected by M. bovis, field studies aiming at the formation of a mycoplasma free herd, were carried out with a group of newborn female calves. These calves were strictly separated from their dams and any other cattle immediately after parturition. Intensive investigations for mycoplasmas were made over 30 months (mycoplasma isolation from nasal swabs, antibody detection by means of indirect hemagglutination test and ELISA technique). M. bovis could never be isolated from the samples. Also, there were no antibodies to M. bovis. In some animals antibody titers to M. bovis occurred after having contact with cattle infected with M. bovis. The results demonstrate a practicable way to establish cattle herds free from M. bovis infection.
    Berliner und Münchener tierärztliche Wochenschrift 08/1992; 105(7):230-2. · 0.82 Impact Factor
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    Evelyn Berthold · M Heller · H Pfützner · R Leirer · K Sachse ·
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    ABSTRACT: Monoclonal antibodies (mabs) against Mycoplasma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas actively secreting mabs against M. bovis, 6 stable lines were cloned. Two of them, Mb 5D8 and Mb 4F6, recognized M. bovis antigens of estimated molecular weights of 33 and 26 kDa, respectively. They showed no cross-reaction to other bovine mycoplasmas, thus rendering them useful for specific detection of this pathogen. All mabs investigated cross-reacted with M. agalactiae which is known to be closely related to M. bovis, but does not occur in cattle. Two other mabs, Mb 5D4 and Mb 1F6, exhibited further cross-reactions to a number of bovine mycoplasma species. Finally, mabs Mb 5D5 and Mb 2G5 reacted with all mycoplasmas tested. The possibility that they recognized constituents of the broth culture medium is discussed. Monoklonale Antikörper (mAk) gegen Mycoplasma (M.) bovis, einen bedeutenden Mastitiserreger des Rindes, wurden mit dem Ziel der Anwendung in spezifischen diagnostischen Methoden hergestellt. 6 von ursprünglich 32 Hybridomlinien, die monoklonale Antikörper gegen M. bovis sekretierten, wurden kloniert. Zwei der mAk (Mb 5D8 und Mb 4F6) waren gegen M. bovis-Antigene mit einem Molekulargewicht von ca. 37 bzw. 26 kDa gerichtet. Sie zeigten keine Kreuzreaktionen zu anderen bovinen Mykoplasmen, wodurch sie für die spezifische Diagnose von Mastitiden bei Kühen, hervorgerufen durch M. bovis, geeignet sind. Alle untersuchten mAk reagierten mit M. agalactiae einer zu M. bovis eng verwandten, aber an Ziege und Schaf adaptierten Spezies. Bei zwei der hergestellten mAk (Mb 5D4 und Mb 1F6) traten weitere Kreuzreaktionen zu einigen anderen bovinen Mykoplasmenspezies auf. Die mAk Mb 5D5 und Mb 2G5 reagierten mit allen untersuchten Mykoplasmen. Die Möglichkeit, daß sie gegen Kulturmedienbestandteile gerichtet sind, wird diskutiert.
    Zentralblatt für Veterinärmedizin. Reihe B. Journal of veterinary medicine. Series B 08/1992; 39(5):353-61. DOI:10.1111/j.1439-0450.1992.tb01180.x
  • Horst Pfützner · Peter Otto ·
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    ABSTRACT: A total of 31 primary cell culture preparations of calf kidney including their processing stages (kidney, washing fluid, cell suspension, cell culture monolayer) were investigated for mycoplasmas by cultural methods. Mycoplasma (M.) arginini, was isolated from 8 out of 27 investigated samples of calf kidney from 3 out of 26 washing fluids, 5 out of 20 cell suspensions, and from 9 out of 21 cell culture monolayers. Furthermore, M. arginini was repeatedly found in throat swabs of the cell laboratory technicians. The results give indications as to the source and route of mycoplasma infections of primary cell cultures.
    Zentralblatt für Bakteriologie: international journal of medical microbiology 07/1992; 277(1):49-53. DOI:10.1016/S0934-8840(11)80870-4
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    K Sachse · Christine Grajetzki · H Pfützner · Renate Hass ·
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    ABSTRACT: Whole-cell protein patterns generated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were compared for 34 isolates of Mycoplasma bovis. A high degree of similarity between most of the strains was established with strain-to-strain differences mainly confined to quantitative variations of certain protein bands, particularly in the molecular weight regions of 64-68, 55 and 26 kD. Two of the isolates provided more deviating patterns. Hydrophobic membrane protein fractions of the strains as prepared by Triton X-114 phase partitioning were compared by SDS-PAGE, which confirmed some of the characteristic strain features found with whole-cell proteins. The immunoblot analysis revealed that up to 20 of the 50-55 discrete protein bands detected in SDS-PAGE patterns were recognized to be antigenic by rabbit and bovine hyperimmune sera. It is concluded that the same set of major antigens is present in all strains investigated, but amounts of individual constituents may be differing.
    Zentralblatt für Veterinärmedizin. Reihe B. Journal of veterinary medicine. Series B 07/1992; 39(4):246-52. DOI:10.1111/j.1439-0450.1992.tb01165.x
  • H Bocklisch · Sybille Kreusel · Annette Bryś · H Pfützner ·
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    ABSTRACT: Twelve freshly lactating ewes were experimentally infected with 2 Mycoplasma (M.) bovis strains via the teat canal in the left udder. The M. bovis infection produced a febrile clinical mastitis in all infected animals. M. bovis could be re-isolated regularly from the experimentally infected udder halves and the infection spread to the other halves. Some contact animals and 4 suckling lambs became naturally infected. Antibody titres were detected by means of the indirect hemagglutination test in blood sera 2 to 3 weeks post infectionem. The pathological lesions were similar to those of the M. bovis mastitis of cows. By the end of the trial the ewes had recovered from the clinical mastitis.
    Zentralblatt für Veterinärmedizin. Reihe B. Journal of veterinary medicine. Series B 08/1991; 38(5):385-90. DOI:10.1111/j.1439-0450.1991.tb00886.x
  • A Brys · H Pfützner ·
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    ABSTRACT: Mycoplasma (M.) bovis hyperimmune serum was subcutaneously injected to 16 of 26 calves repeatedly intranasally infected with M. bovis, during and/or after experimental infection. Antibody titres between 1:32 and 1:256 were recorded by means of indirect haemagglutination from the calves treated. Transmission of film-inhibitory antibodies failed to work. Neither clinical manifestations nor pathologico-anatomic alterations to the lungs of experimentally infected animals were mitigated by hyperimmune serum treatment. M. bovis, in high germ counts, was re-isolated from nasal swabs, trachea, pulmonary lymph nodes, and inflammatory lung tissue of both treated and untreated calves.
    Archiv für experimentelle Veterinärmedizin 02/1989; 43(5):677-83.
  • S Kreusel · H Bocklisch · H Pfützner · A Brys · R Leirer · U Ziegenhals ·
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    ABSTRACT: M. bovis or M. bovigenitalium species experimentally used in intrapreputial infection were re-isolated from 7 of 8 bulls. Clinically manifest diseases did not develop at all, though slight inflammatory lesions were recorded from the genital tract of 2 animals. Mycoplasma findings are discussed together with results obtained from spermatological and hematological investigations.
    Archiv für experimentelle Veterinärmedizin 02/1989; 43(5):705-12.
  • A I Sobko · V D Nastenko · V P Berdnik · D Schimmel · H Pfützner ·
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    ABSTRACT: Four experimental series were run in 2 experiments with 44 unweaned piglets to test non-inactivated vaccine from ts-mutant M-60 of Mycoplasma (M.) arginini and from attenuated strains of CH-2 M. hyorhinis, EP-29 M. hyosynoviae, M. suipneumoniae, and B-1 Acholeplasma laidlawii. Similar deviations of clinical and immunological parameters were recorded from piglets inoculated with the above vaccine and infected with pathogenic mycoplasma cultures. These deviations, however, were less strongly pronounced in animals which had been inoculated. Mycoplasma species were re-isolated from bronchial lymph nodes and lungs of 62.5% of inoculated piglets. Lasting residual virulence was recorded from the attenuated mycoplasma strains. That residual virulence had no substantial impact upon growth and development of the piglets under laboratory conditions, throughout the period of observation. The above results are likely to suggest the advisability of further studies for the development of a vaccine from ts-mutants and attenuated strains of pathogens of mycoplasmosis in swine.
    Archiv für experimentelle Veterinärmedizin 02/1989; 43(5):645-55.
  • H Bocklisch · H Pfützner · V Zepezauer ·
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    ABSTRACT: Mycoplasma (M.) ovipneumoniae was isolated pure or mixed with bacteria from 47 lungs of lambs of 14 in 22 tested flocks. M. ovipneumoniae was obtained as pure culture in cases of mild bronchopneumonia. Experimental intratracheal or intranasal infection caused several days of rising body temperature above 39.7 degrees C. Nasal discharge, coughing, and dyspnea did not occur. M. ovipneumoniae was successfully re-isolated from nasal swabs, beginning 2 d from infection. Lobular catarrhal bronchopneumonia was established by postmortem examinations, 10-14 d from infection, and M. ovipneumoniae was re-isolated from the lungs. Histological patterns of lungs were characterised by interstitial cell reactions.
    Archiv für experimentelle Veterinärmedizin 02/1989; 43(5):755-61.
  • H Bocklisch · V Zepezauer · H Pfützner · S Kreusel ·

    Archiv für experimentelle Veterinärmedizin 04/1987; 41(2):249-57.
  • H Pfützner · C Wehnert · R Leirer ·

    Archiv für experimentelle Veterinärmedizin 02/1986; 40(1):56-62.
  • C Wehnert · H Pfützner ·

    Archiv für experimentelle Veterinärmedizin 02/1986; 40(1):75-81.