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.
Project lay out identified the following tasks:
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.
improve biochemical testing procedures used to distinguish MmmSC
from other members of the Mycoplasma mycoides cluster. To develop
easy and rapid biochemical tests.
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.
immunity (CMI) tests such as gamma interferon (gINF) and skin
tests were thought to be a contribution to the development of
improved diagnostic methods.
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.
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
had the aim of infecting cattle and sheep under controlled conditions
to make specimens available for the evaluation of the novel
VIII-Testing of cattle affected by pulmonary mycoplasmosis.
originating from Hungarian cattle affected by pulmonary mycoplasmosis
were tested, to assess specificity of the testing systems under
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.
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
novel IBT testing system identifies the carrier state in chronically
infected animals and false positive reactors to CFT.
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.
Enzyme-linked immunofilter assay (ELIFA) was successfully applied
to detection of MmmSC in clinical specimens
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.
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
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.
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.
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
VIII- Testing of Hungarian cattle affected by pulmonary mycoplasmosis
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.
Zooprofilattico Sperimentale dell Abruzzo e del Molise G. Caporale
+39 086 133 22 28
+39 086 133 22 51
- Robin A.J. NICHOLAS
Central Veterinary Laboratory
UK-KT15 3NB New Haw - Addlestone
Tel.: +44 932 35 73 79
Fax: +44 932 35 75 95
- Karl-Erik JOHANSSON
National Veterinary Institute
Ulls Vâg 2B
P.O. Box 7073
S-750 07 Uppsala
Tel.: +46 18 67 40 00
Fax: +46 18 30 91 62
- Roger Joseph MILES
University of London
King's College London
Campden Hill Road
UK-W8 7AH London
Tel.: +44 1713 33 44 11
Fax: +44 1713 33 45 00
- Josè REGALLA
Instituto de Protecção da Produção
Estrada de Benfica 701
Tel.: +351 21 716 20 75
Fax: +351 21 716 00 39
- Fulgencio GARRIDO ABELLAN
Ministerio de Agricultura, Pesca y Alimentacion
Camino de El Jau
E-18320 Santa Fe (Granada)
Tel.: +34 95 844 03 75
Fax: +34 95 844 12 00
- Laszlo STIPKOVITS
Veterinary Medical Research Institute
Hungária krt 21
HU-1143 Budapest XIV
Tel: +36 1 252 10 69
Fax: +36 1 252 24 55