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Development of new and improved diagnostic tests for contagious bovine pleuropneumonia in Europe (CBPP)

Contract nr: FAIR-CT95-0711
Project nr: 711
Project type: SC
Starting date: 01/03/1996
Duration: 36 months
Completion date: 30 April 1999
Total cost: 1,566,600 EUR
EC Contribution: 823,100 EUR
Scientific Officer: Isabel MINGUEZ-TUDELA
Research topic: Animal health
Acronym: CBPP Diagnosies and Research

Contagious bovine pleuropneumonia (CBPP) is an eluding pathology, and can go undetected for months. This state of affairs hampers progress in disease control and eradication. The disease is still present in the Iberian Peninsula and doubts exist about its presence in Eastern Europe. Furthermore, CBPP is spreading through the African continent, where it is a major obstacle to livestock development.

The Project lay out identified the following tasks:

Task I-Culture Media
Mycoplasma mycoides subsp. mycoides small colony (MmmSC) is a fastidious organism to grow. The objective was to develop and optimise transport and growth media and minimise batch variability.
Task II-Biochemistry
To improve biochemical testing procedures used to distinguish MmmSC from other members of the Mycoplasma mycoides cluster. To develop easy and rapid biochemical tests.
Task III-Serology
Available tests for detection of circulating antibody lack specificity and sensitivity. The aim was to harmonise and evaluate Immunoblotting (IBT) as a serological diagnostic test, to produce monoclonal antibodies to be used in assays such as ELISA, IBT and immunocytochemistry (ICC), and finally, to attempt development of a field test. IBT was thought to be the appropriate system for identification of immunodominant MmmSC cell components, and could also be used for diagnostic purposes.
Task IV-Cellular Immunology
Cell-mediated immunity (CMI) tests such as gamma interferon (gINF) and skin tests were thought to be a contribution to the development of improved diagnostic methods.
Task V-DNA Amplification
Polymerase chain reaction (PCR) suitable for mass screening, based on an enzyme assay rather than gel electrophoresis, was evaluated. The development of an in situ PCR was also investigated.
Task VI-RNA Analysis
A PCR innovative systems for identification and differentiation of members of the Mycoplasma mycoides cluster by genotyping methods of analysis of low molecular weight tRNA profiles was investigated.
Task VII-Animal Studies
This had the aim of infecting cattle and sheep under controlled conditions to make specimens available for the evaluation of the novel diagnostic systems.
Task VIII-Testing of cattle affected by pulmonary mycoplasmosis.
Specimens originating from Hungarian cattle affected by pulmonary mycoplasmosis were tested, to assess specificity of the testing systems under development.

Current situation/Results:

Task I-Culture Media
A complex medium (PRM) for MmmSC, giving high growth rates and cell yields, was formulated. Other media developed included a highly selective transport medium and a semi-defined medium.
Task II-Biochemistry
The patterns and kinetics of substrate utilisation by SC strains were determined. It was shown that European SC strains are unable to oxidise glycerol, whereas glycerol was oxidised by African and Australian SC isolates, and by all other strains of M. mycoides. A glycerol oxidation test was developed. Furthermore, maltose-utilising ability was shown to be a major feature distinguishing SC strains (negative) from other members of the M. mycoides cluster. A 30 min. maltose colour test was also developed
Task III-Serology
The novel IBT testing system identifies the carrier state in chronically infected animals and false positive reactors to CFT.
A novel, indirect ELISA and a rapid latex agglutination test (LAT) were developed. The latter assay can be used by field personnel. A monoclonal antibody raised against MmmSC was found to be useful for the specific immunochemical detection of antigen in formalin-fixed tissue.
An Enzyme-linked immunofilter assay (ELIFA) was successfully applied to detection of MmmSC in clinical specimens
Task IV-Cellular Immunology
A gINF test for detection of MmmSC infected animals was set up, but further work is required to assess its specificity and sensitivity. A delayed hypersensitivity test (DHT) gave inconclusive results. A mini-kit IBT system, to facilitate standardised performance of the test in diagnostic laboratories, has been made available.
Task V-DNA Amplification
The enzymatic PCR colorimetric assay for screening simultaneously large numbers of specimens was found to be an appropriate diagnostic tool. In situ PCR was also developed. It can be used in parallel with ICC
Task VI-RNA Analysis
The 16S rRNA gene sequences of the rrnA and the rrnB operons have been determined for European, Australian and African strains of MmmSC. A PCR system based on the 16S rRNA genes of MmmSC was found to be sensitive and applicable to samples from affected cattle.
A distinction between MmmSC and MmmLC was achieved by tRNA profile analysis. The system proved to be a useful complement or alternative to conventional methods for identification of new isolates. There was a reproducible and significant species-to-species variation in the tRNA profiles obtained, but no variations were seen between MmmSC field isolates.
Task VII-Animal Studies
Infection of cattle, under controlled conditions, provided a further opportunity to assess the performance of the newly developed diagnostic systems. PCR was confirmed to be an extremely sensitive diagnostic testing system.
Task VIII- Testing of Hungarian cattle affected by pulmonary mycoplasmosis
Specimens collected from abattoirs and/or farms were examined by bacteriological, serological and PCR tests. No MmmSC or antibodies were detected in any instance. Pulmonary pathologies were due, in many cases, to infection by M. bovis.


John BASHIRUDDIN (till March 1999) thereafter Attilio PINI
Istituto Zooprofilattico Sperimentale dell Abruzzo e del Molise G. Caporale
Via Campo Boario
I- 64100 Teramo
Tel.: +39 086 133 22 28
Fax: +39 086 133 22 51


  • Robin A.J. NICHOLAS
    Central Veterinary Laboratory
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  • Karl-Erik JOHANSSON
    National Veterinary Institute
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  • Roger Joseph MILES
    University of London
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  • Josè REGALLA
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    Veterinary Medical Research Institute
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