Download Disease name

OIE Reference Laboratory Reports
Activities in 2010
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory
Contagious bovine pleuropneumonia
(CBPP)
INRB, I.P – LNIV - Laboratório
Nacional de Investigação Veterinária
Estrada de Benfica 701, 1549-011 Lisboa, Portugal
Tel.:
(+351-21) 711.53.39
Fax:
(+351-21) 711.52.36
e-mail address:
[email protected]
website:
Name of Head of Laboratory
(Responsible Official):
Name of OIE Reference Expert:
Name of writer of this report
(if different from above):
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
http://www.inrb.pt/
Nuno Canada
Ana Botelho
---------
1
Contagious bovine pleuropneumonia
Part I: Summary of general activities related to the disease
1.
Test(s) in use/or available for the specified disease/topic at your laboratory
The complement fixation test (CFT), a modified method of Campbell and Turner, is the international reference test
recommended by OIE, for diagnosis of Contagious Bovine Pleuropneumonia (CBPP). A serobank of more than
1000 sera from naturally infected and few from experimentally infected cattle is under maintenance and
organization, what demands a full register of each sera and evaluation and confirmation of its titre by CFT.
The immunobloting test (IBT), developed in LNIV and recommended by OIE as a confirmatory test, is routinely
performed to positive or doubtful CFT sera to confirm the results due to the possibility of false positives due to
cross-reactions. From all the sera received in 2010 that gave positive or doubtful results in CFT, none was
confirmed by IBT to be CBPP positive. This fact has been verified in the last seven years and, therefore, the CBPP
surveillance plan changed for 2011, according to the National Veterinary Authority – DGV, and CFT serological
screening test will be reduced and surveillance will be done mainly by clinical signs and by suspected lesions at
the abattoir.
Polymerase chain reaction (PCR and PCR-REA) allows the rapid, specific and sensitive detection and
identification of the aetiological agent of CBPP Mycoplasma mycoides subsp. mycoides small colony (SC) in
clinical material and in culture. As the last case of CBPP in Portugal was reported in 1999 and in 2003 the country
obtain the CBPP free status certificate from OIE, no Mycoplasma mycoides subsp. mycoides SC strain has been
isolated since then and the few PCR tests performed, for the diagnostic of the disease, have been all negative.
Test
Specificity
Maintenance and
evaluation of CBPP
positive sero-bank
Surveillance
Research
Total
Serological
assay CFT
Detection of
Mycoplasma spp,
of the
Mycoplasma
mycoides group,
antibodies
782
13*
–
795
Serological
confirmatory
assay IBT
Detection of
Mmm SC specific
antibodies
–
1068
–
1068
Molecular test PCR and PCRREA
Detection of
Mmm SC DNA in
samples and
identification of
isolates
–
6
81
87
* The CBPP screening of herds by CFT is mainly performed at Regional Veterinary Laboratories,
officially approved and trained by the NRL – LNIV to perform this test.
2.
Production and distribution of diagnostic reagents
The two European standard bovine Positive Sera, mentioned in OIE Manual 2004, PS1 and PS2, prepared at LNIV
from Portuguese CBPP naturally infected bovine sera, for antigen titration and CFT harmonisation purposes, have
been supplied to several institutions. Other CFT reagents were also supplied as follows:
2
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Contagious bovine pleuropneumonia
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied to
other countries*
Mmm SC Antigen for CFT and
IBT tests
Two batches of 125 ml each (total 250 ml –
50 flasks – 5 ml each)
50 ml
Positive reference bovine sera
(PS1 and PS2)
47 ml (distributed in 1 ml)
5 ml
Negative reference bovine sera
44 ml (distributed in 1 ml)
3 ml
* South Africa, China and Romania
Part II: Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
None
4.
5.
Preparation and supply of international reference standards for diagnostic tests or vaccines
Reference Material
Amount supplied to other OIE Members*
Mmm SC Antigen for CFT
50 ml
CBPP Positive reference bovine sera (PS1 and PS2)
5 ml
Negative reference bovine sera
3 ml
Research and development of new procedures for diagnosis and control
The following project is in progress:
Development of improved Nucleic Acid Test (NAT) kits for detecting MmmSC and M. bovis in bovine samples.
One important challenge regards the assembly of user-friendly and field-deployable assays, which should be
independent of the utilization of sophisticated equipments and adequate for a wider use in low-resource
laboratories and countries. Isothermal DNA amplification and lateral flow chromatography will be explored for
this purpose. Real-time PCR-based assays will be also developed for use in better-equipped laboratories.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
None
7.
Provision of consultant expertise to OIE or to OIE Members
OIE expert mission to the People’s Republic of China: Evaluation of Contagious Bovine Pleuropneumonia
(CBPP) free status
Participation in an international mission to People’s Republic of China, to verify, based on the terms of reference,
if the CBPP free status can be attributed according to OIE standards by clarifying some aspects of the dossier
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
3
Contagious bovine pleuropneumonia
submitted by PR of China. Two OIE experts participated: Herbert Schneider and Ana Botelho, from 14 to
27 November 2010, and had meetings at the MOA level and province level and visited the reference laboratory in
Harbin and regional laboratories and abattoirs in two provinces. A detailed report was presented to OIE to be
analysed by the CBPP ad hoc group and no follow up was considered necessary.
8.
Provision of scientific and technical training to personnel from other OIE Members
None
9.
Provision of diagnostic testing facilities to other OIE Members
None
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
11. Participation in international scientific collaborative studies
None
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)

Presentations at international conferences and meetings
Second Global Conference of OIE Reference Laboratories and Collaborating Centres, OIE Headquarters, Paris,
France, 21-23 June 2010. A leaflet was prepared: “VNTR analysis in the epidemiological study of infectious
diseases: a retrospective evaluation of CBPP in Portugal “

Scientific publications in peer-reviewed journals
Varela, F., Inácio, J. & Botelho, A. 2010. Molecular diversity assessed by VNTR and IS1296 typing of historical
Mycoplasma mycoides subsp. mycoides SC strains. Veterinary Microbiology 146: 295–302
The last case of Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides
SC (MmmSC), in Europe was reported in Portugal in 1999. However, in view of its insidious nature, it is still
possible that CBPP could re-emerge. Despite differences in animal host and geographical origin, most of the
European MmmSC field isolates were traditionally considered to be very homogeneous. In the present study we
performed a retrospective variable-number tandem repeat (VNTR) and IS1296 genotyping analysis of 65 MmmSC
field isolates associated to the last CBPP outbreaks that occurred in Portugal in order to elucidate their intraspecific genetic variability. A 8.8 kb region and two VNTR loci (VNTR4 and VNTR5) were analyzed for
polymorphisms by PCR amplification. All but one strain presented the same IS1296 profile, in contrast with the
VNTR genotyping that confirmed some diversity of Portuguese strains showing VNTR4, the most discriminatory
one, four different patterns. VNTR4 type ‘‘9’’ (numbering according to the estimated number of repeats) was the
most predominant one mainly in the Entre Douro–Minho region. All isolates from one geographic region (Beira
Litoral) presented VNTR4 type ‘‘8’’ suggesting the existence of a region-specific VNTR. These facts raise
thehypothesis that at least two CBPP re-emergence events could have occurred in Portugal since 1983 after
30 years of silence. This aspect represents a major concern and is a major reason for the maintenance of intensive
research on this disease.

Other communications
Poster presentation at the I Jornadas Saúde Pública Veterinária, UTAD (Universidade de Trás os Montes e Alto
Douro), Vila Real, Portugal, May 29:
4
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Contagious bovine pleuropneumonia
Varela, F., Inácio, J. & Botelho, A. 2010. Análise de VNTRs no estudo epidemiológico de doenças infecciosas
animais: avaliação retrospectiva da Peripneumonia Contagiosa Bovina em Portugal.
Master Thesis: F. Varela. 2010. Tipificação de Mycoplasma mycoides subsp. mycoides SC e detecção da sua
aderência a células epiteliais pulmonares. New University of Lisbon, FCT. 82 pp.

Abstract
The contagious bovine pleuropneumonia (CBPP) is an infectious respiratory disease of great importance in the
cattle production industry, caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC). Currently, CBPP
remains endemic in Africa, where it is responsible for important losses in the livestock sector. The last outbreak of
CBPP in Europe occurred in Portugal in 1999 but, due to its insidious nature, the risk of re-emergence of this
disease is permanent. MmmSC strains associated with the recent CBPP outbreaks in Europe have been considered
genetically very homogeneous. However, recent molecular typing studies revealed the existence of an intraspecific
genetic variability higher than originally expected. This work was based on two fundamental objectives: the typing
of MmmSC strains isolated during the last CBPP outbreaks that occurred in Portugal between 1993 and 1998, and
the implementation of an alternative and innovative method for the detection and quantification of MmmSC after
infection of cell cultures. To achieve the first objective we analised the variability of polymorphic VNTR
(Variable Number of Tandem Repeats) regions existing in the genome of MmmSC strains. The locus VNTR4
proved to be the most polymorphic, presenting four distinct profiles among the Portuguese strains. The type "9"
VNTR4 profile (called according to the number of repetitions of the consensus sequence) proved to be widely
distributed and was predominant among the isolates. However, there was a geographical segregation of profiles,
since all strains found in the region of Beira Litoral presented a type "8" VNTR profile. These results suggest that
at least two CBPP re-emergence events may have occurred in Portugal between 1993 and 1998. To achieve the
second objective, a fluorescence in situ hybridisation (FISH) technique was optimized that allowed the direct
visualization of mycoplasmas adherent to cell cultures. This technique, which has never been applied previously
for the detection of MmmSC, proved to be adequate for future applications in cytadherence studies and for the
detection of mycoplasmas in cell cultures.
Keywords: Contagious Bovine Pleuropneumonia (CBPP), Mycoplasma mycoides subsp. mycoides SC, VNTR
(Variable Number of Tandem Repeats), FISH (Fluorescence In Situ Hybridisation)
13.
Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
No
ii)
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
which kits?
No
_______________
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
5