Brucellosis
Mrs J A Stack
Veterinary Laboratories Agency, Weybridge
New Haw, Addlestone, Surrey, KT15 3NB, United Kingdom
Tel: (+44-1932) 35.76.10, Fax: (+44-1932) 35.72.16
j.a.stack@vla.defra.gsi.gov.uk - www.defra.gov.uk/corporate/vla
Summary of general activities related to the disease
1.
Test(s) in use/or available for the specified disease at your laboratory
a) Serology
Diagnostic tests were performed for surveillance, diagnosis and international trade.
b)
Test
For
Import/Export
Surveillance
Total
RBT
Smooth Brucella
167
2037
2204
iELISA
Smooth Brucella
4361
404723
409084
cELISA
Smooth Brucella
2669
32795
35464
SAT
Smooth Brucella
4629
4510
9139
CFT
Smooth Brucella
3766
3715
7481
SAT
B. canis
2132
0
2132
RSA
B. canis
983
0
983
CFT
B. ovis
296
0
296
Milk
Standardised iELISA reagents were provided by this laboratory for the 170,000 bulk tank milk samples tested by
commercial laboratories. This laboratory provides confirmatory testing of positive milk samples, follow up blood
tests and quality assurance monitoring.
c)
Culture
Culture and identification of Brucella using classical and molecular tests were carried out for domestic diagnosis
in animals and humans and as a service provided worldwide. A total of 3354 bovine abortion/calving samples and
11254 samples from sheep (11009) and goats (245) were examined for domestic surveillance purposes.
Sixty five cultures have been submitted from around the world, 45 of which all have been confirmed as Brucella,
typed to biovar level and usually by PCR.
Investigations into marine mammal infections continued, isolations from twelve animals recovered from Scottish
coastal waters were confirmed as Brucella. In addition, tissues from 63 sea mammals were received from the
southern coastal waters of the UK. Brucella was recovered from one Bottlenose Dolphin, seven were serologically
positive.
Eighteen isolates were received from humans in UK and ten were confirmed as Brucella. All had traveled abroad
in the previous twelve months (where history was available).
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Brucellosis
2.
Production and distribution of diagnostic reagents
Reagents were distributed on a commercial and non-commercial basis within the UK and world-wide. We were
invited to present ‘standardisation of reagents at VLA for the detection of brucellosis’ by Istituto Zooprofilattico
Sperimentale dell’Abruzzo e del Molise, Palazzo Trecchi, Italy.
Product
Quantity
MRT antigen (B. melitensis/abortus/suis)
1200ml
SAT antigen (B. melitensis/abortus/suis)
4500ml
RBT antigen (B. melitensis/abortus/suis)
3200ml
RBT antigen (ovine, caprine use) (B.melitensis/abortus/suis)
1200ml
CFT antigen (B. melitensis/abortus/suis)
9500ml
CFT B.ovis antigen
70ml
SAT antigen ( B.canis)
180ml
RSA antigen (B.canis)
40ml
Various phages
35ml
ELISA kits
170
Various antisera
400ml
Monospecific Sera
73
Brucella strains
21
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
We contributed to the first brucellosis serology proficiency ring trial for tests used on cattle, organised by the
Community Reference Laboratory, AFSSA, Paris.
The European ring trial aiming at improvement in the phenotypic & molecular typing of Brucella suis progressed
and is continuing into 2008. samples of DNA from our collection have been sent to the German NRL for
distribution to laboratories who are participating in the VNTR ring trial.
We organised a validation study for milk ELISA. Collaboration with CRL France, NRL Argentina, NRL Portugal
(Azores Islands), IZS Sicily and University of Nigeria should produce validation data from infected animals for
the VLA ELISA kit.
To ensure reliable results and products, VLA operate a Quality Assurance scheme for internal and external assay
confirmation. We incorporate additional applicants, not solely VLA, including those from E.europe, Slovakia,
S. Africa and Singapore in our network of international laboratories.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines.
The candidate International Standard Serum for caprine & ovine brucellosis has been tested amongst the OIE
Reference Laboratories. Results will be available for presentation by VLA to the OIE in 2008.
The collaborative testing of the proposed porcine International Standard Serum on behalf of Dr K. Nielsen (ADRI
Canada) has continued this year.
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Brucellosis
We have supplied the following International Standard sera: 9 ampoules of OIEISS, 6 ampoules of Brucella ovis
International Standards and 5 sets (15) of OIEELISA Standards. 80 International Standard sera have been
distributed to the Community reference Laboratory, AFSSA, France.
5.
Research and development of new procedures for diagnosis and control
The group have continued to develop novel molecular diagnostic techniques. This year we published details of a
multilocus typing scheme [MLST] (Whatmore et al., 2007) examining nine distinct gene fragments from some
160 isolates. We have since extended this work to examine some 21 gene fragments from ~400 isolates and used
this phylogenetic framework to identify valuable diagnostic markers in the form of SNPs that can be incorporated
into diagnostic assays to type rapidly to the level of species and below. A rapid speciation assay based on primer
extension procedures was described this year (Scott et al., 2007) and a manuscript describing a similar assay based
on a potentially more widely applicable real-time PCR platform was submitted for publication towards the end of
the year. We continue to expand our VNTR typing database and have carried out extensive work during the last
year in Northern Ireland to examine the value of the tool in disease control. We hope to report on this within 2008.
Both VNTR and MLST techniques were applied to studies examining relationships between Brucella isolated
from marine mammals (Groussaud et al., 2007) highlighting the value of these approaches as tools in Brucella
molecular epidemiology. The group have continued to work on the development of novel and improved
immunodiagnostic techniques, and in particular the evaluation of novel Brucella specific proteins for use in
serological and cellular response assays. Alternative, improved sensitivity assays for both serological and cell
mediated effectors have been generated and evaluation with stored field material is currently underway. The
assays show promise for the discrimination of false positive serological reactions and also for the discrimination of
vaccinated animals from infected animals. We continue to collect sera and plasma samples from worldwide
collaborators for validation of these novel assays, and the provision of such material is essential for the success of
this work. Work on vaccine development continues to focus on the development of novel sub-unit vaccines (nonliving) for brucellosis. Initial vaccine development work, describing the identification of two protective DNA
vaccine candidates, was published in 2007 (Commander et al 2007).
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
The recent valid publication of two new species B. ceti sp. nov. and B. pinnipedialis sp. nov. proposed as names of
isolates from cetaceans and seals (Foster. G., et al 2007) is controversial and has hightened the need to resolve the
issue. The grouping of strains in a recent study (Dawson. C., et al in preparation) shows remarkable agreement
with the results of some of the same strains tested in another study (Bourg, G., et al 2007). Further data arising
from recent studies from both Variable-Number-of Tandem-Repeats (VNTR) analysis and multilocus sequence
analysis have revealed strains from these species can also be divided into the same three groups (Groussaud, P., et
al 2007). The data presented in these studies appears to be more compatible with proposals outlined by Corbel &
Bani (Bergey’s Manual of Systematic Bacteriology 2005) in which the possibility of identification of three novel
species ‘Brucella phocae’ (seals), ‘Brucella phoecoenae’ (porpoises) and ‘Brucella delphini’ (dolphins), was
raised.
Using our range of molecular tools we have become aware of novel strains of Brucella. This includes an isolation
from a human breast implant, the confirmation was requested during collaboration with CDC. Additionally we
were asked to examine a Brucella strain from a baboon from the SW biomedical Research Institute in Texas which
has also shown to be dissimilar to classical strains. We have discovered an unusual isolate from a vole at the
request of the German Medical Laboratory and also a marine mammal Brucella genotype associated with zoonotic
infection. All these findings are due for publication in 2008.
7.
Provision of consultant expertise to OIE or to OIE Member Countries
We were asked to perform initial assessment of National laboratory of Veterinary investigation (LNIV) Portugal to
evaluate Brucella tests by Portuguese Accreditation Institute according to EN ISO 17025:2005.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
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Brucellosis
8.
Provision of scientific and technical training to personnel from other OIE Member Countries
We hosted short term visitors from USA, Sweden, Korea and Brazil. No practical training was performed.
9.
Provision of diagnostic testing facilities to other OIE Member Countries
We have confirmed isolates from UAE, Italy, Turkey, Sicily, France, Korea, Albania and India.
We have received sera from Cyprus, Sicily, Thailand, Croatia, Canada, Slovakia and Tajikistan.
We examined a CFT kit prepared by Istituto Superiore di Sanita, Rome using the OIE B. ovis standard antisera.
VLA opinion was sought regarding serological positive heifers exported from Sweden to Lithuania. VLA tests
agreed with Lithuania, some were serologically positive- currently there are no tests that can distinguish these
false reactions from those of true reactors.
Clarification of milk assays for Riyadh was provided to assist in a situation where 50,000 brucellosis free milking
cows had been vaccinated with Rev-1 and were giving MRT reactions, which were also positive to Svanova
cELISA & RBT. We offered to draw up a strategy for vaccination & surveillance and to attempt isolation, PCR
and typing for them as this is the only way of identifying vaccination from infection. Investigations continue and a
preliminary study of cross reactivity of Svanova cELISA kit is underway.
10. Organisation of international scientific meetings on behalf of OIE or other international bodies.
None
11. Participation in international scientific collaborative studies
Following a visit to VLA by Dr. Donghee Cho from The National Veterinary Research Quarantine Service, Gyeonggi,
Republic of Korea we have a formal collaborative agreement. The primary role is for support and harmonisation,
there will be joint activities and meetings between scientists with workshops and short-term placements in each
other’s laboratories. We will encourage the development of joint proposals for research funding. We are proposing
this lab in Korea becomes twinned with us as a OIE NRL.
Ad hoc advice was frequently sought. For example, to assist in making diagnoses, develop diagnostic capability,
boisafety or technical aspects to Bosnia and Herzegovina, Croatia, Egypt, Japan, Malaysia, India, Pakistan, Poland,
Sweden and Turkey.
Collaborations with Albania continue regarding the vaccine strategy there and the kind provision of samples for
research to VLA. We have formally provided advice to Afghanistan (EU funded project) regarding procedures and
setting up the laboratory and recent collaboration with NRL Poland has led to the exchange of reagents and a
proposed technical training visit to VLA in 2008.
As a FAO Collaborating centre, we are frequently approached by the FAO to assist in countries such as Armenia
Azerbaijan, Tanzania, Tajikistan, and this year. This involves consultancy, lab workshop/design, provision of
reagents, technical advice, methodology, control strategies etc
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
Gopaul, K.K., Smith, C.J. and Whatmore, A.M. The development of SNP discrimination assays on a real-time
PCR platform for rapid discrimination of Brucella at the species level. 60th Annual Brucellosis Research
Conference, Chicago IL, USA, 1-2nd December 2007.
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Brucellosis
Whatmore, A.M., Sawson, C., Groussaud, P., Koylass, M.S., ing, A., Shankster, S.J., Sohn, A.H., Probert,
W.S., McDonald, W.L. A marine mammal Brucella genotype associated with zoonotic infection. 60th Annual
Brucellosis Research Conference, Chicago USA, 1-2nd December 2007.
Stack, J.A., Perrett, L.L., Groussaud, P. and Whatmore, A.M. Brucellosis – Classification of human isolates in
Great Britain. 5th International Conference on Emerging Zoonoses, Limassol, Cyprus, 15-18th November 2007.
Kopaul, K.K., Smith, C.J. and Whatmore, A.M. The development of SNP discrimination assays on a real-time
PCR platform as a tool for the rapid identification and speciation of Brucella. 13th International World Association
of Veterinary Laboratory Diagnosticians Symposium (WAVLD), Melbourne, Australia, 11-14th November 2007.
Stubberfield, E., Dawson, C., Perrett, L., Brew, S. and Stack, J. Brucellosis in marine mammals: Veterinary
Laboratories Agency involvement. 13th International World Association of Veterinary Laboratory Diagnosticians
Symposium (WAVLD), Melbourne, Australia, 11-14th November 2007.
Commander, N and Perkins, S. Development of animal and human vaccines against brucellosis. Zoonoses:
From science to policy (International conference), Thistle Hotel, Glasgow, UK, 5-7th November 2007.
Perrett, L.L., Groussaud, P., Whatmore, A.M., Brew, S.D. and Stack, J.A. Characterisation of human
brucellosis in the UK. Zoonoses: From science to policy (International conference), Glasgow, UK, 5-7th
November 2007.
Stack, J.A. Brucellosis activities at VLA and current research projects. Presented at update meeting, InBios,
Seattle, Washington, USA, 15th October 2007.
Commander, N. and Thirlwall, R. Discrimination of vaccinated animals from infected animals using serology.
Istituto Zooprofilattico Sperimentale della Sicilia (ISZS), Palermo, Sicily, Italy, 21-22nd September 2007.
Perrett, L. and Stack, J. Biotyping – The Gold Standard. Istituto Zooprofilattico Sperimentale della Sicilia
(ISZS), Palermo, Sicily, Italy, 21-22nd September 2007.
Stack, J. Brucellosis. Istituto Zooprofilattico Sperimentale della Sicilia (ISZS), Palermo, Sicily, Italy, 21-22nd
September 2007.
Whatmore, A. Molecular detection and typing of Brucella at the VLA. Istituto Zooprofilattico Sperimentale della
Sicilia (ISZS), Palermo, Sicily, Italy, 21-22nd September 2007.
Koylass, M.S., Stubberfield, M., Scott, J. and Whatmore, A. Using multilocus sequence typing to develop a
primer extension assay based on single nucleotide polymorphisms (SNP) for typing Brucella spp. 107th General
Meeting American Society for Microbiology, Toronto, Canada, 21-25th May 2007.
Gopaul, K.K., Smith, C.J. and Whatmore. Development of a Real-Time PCR tool for speciation within the
genus Brucella. 107th General Meeting American Society for Microbiology, Toronto, Canada, 21-25th May 2007.
Stack, J.A. The role of the Veterinary Laboratories Agency (Weybridge) as an OIE reference center for
brucellosis. 15th International Congress of Mediterranean Federation for Health and Production of Ruminants,
Kusadasi, Turkey, 15-19th May 2007.
Commander, N.J. Novel Brucella vaccines and development in diagnostic tests for differentiating field infection
from vaccination. 15th International Congress of Mediterranean Federation for Health and Production of
Ruminants, Kusadasi, Turkey, 15-19th May 2007
Stubberfield, E., Dawson, C., Perrett, L., Brew, S., Whatmore, A., Groussaud, P., King, A. and Stack, J.
Brucellosis in marine mammals: Veterinary Laboratories Agency involvement. 38th Annual conference
International Association for Aquatic Animal Medicine (IAAAM), Lake Buena Vista, Florida, 5-9th May 2007.
Taylor, A. Serological reagents for the detection of brucellosis. The Veterinary Laboratories Agency: Working for
public and animal health, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Cremona, Palazzo
Trecchi, Italy, 16th March 2007.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007
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Brucellosis

Scientific publications in peer-reviewed journals
Commander, N.J., Spencer, S.A., Wren, B.W. and MacMillan, A.P. The identification of two protective DNA
vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes. (2007) Vaccine, 25(1):
43-54.
Groussaud, P., Shankster, S.J., Koylass, M.S., and Whatmore, A.M. Molecular typing divides marine mammal
strains of Brucella into at least three groups with distinct host preferences. (2007) Journal of Medical
Microbiology, 56: 1512-1518.
Jackson, R., Ward, D., Kennard, R., Amirbekov, M., Stack, J., Amanfu, W., El-Idrissi, A. and Otto, H.
Survey of the seroprevalence of brucellosis in ruminants in Tajikistan. (2007) Vet. Rec., 161(14): 476-482.
McGiven, J., Hendry, L., Brown, D., Gillard, K., Katiyar, A., Stack, J., Perrett, L., Rollins, M., Hughes, S.,
Ready, R., Kemp, P., Morris, P. and Mawhinney, I. The development of an efficient test automation and
sample tracking system in a high throughput veterinary laboratory to improve the emergency response to
outbreaks of highly infectious disease in Great Britain. (2007) JALA, 12(2):73-82.
Minas, A., Stournara, A., Minas, M., Stack, J., Petridou, E., Christodoulopoulos, G. & Krikelis, V.
Validation of fluorescence polarization assay (FPA) performed in microplates and comparison with other tests
used for diagnosing B.melitensis infection in sheep and goats. (2007) Journal of Immunological Methods, 320(12): 94-103.
Scott, J.C., Koylass, M.S., Stubberfield, M.R. and Whatmore, A.M. Multiplex assay based on singlenucleotide polymorphisms for rapid identification of Brucella isolates at the species level. (2007) Applied and
Environmental Microbiology, 73(22): 7331-7337.
Stournara, A., Minas, A., Bourtzi-Chatzopoulou, E., Stack, J., Koptopoulos, G., Petridou, E. and Sarris, K.
Assessment of serological response of young and adult sheep in conjunctival vaccination with Rev-1 vaccine by
fluorescence polarization assay (FPA) and other serological tests for B.melitensis. (2007) Veterinary
Microbiology, 119(1): 53-64.
Whatmore, A.M., Perrett, L.L., and MacMillan, A.P. Characterisation of the genetic diversity of Brucella by
multilocus sequencing. (2007) BMC Microbiology, 7:34.

Other communications
Renewed application to remain FAO Reference Laboratory for brucellosis
Renewed designation as the WHO Collaborating Centre for Reference & Research on Brucellosis
Participated in 2007 WHO Stakeholders perceptions Questionnaire conducted by GlobeScan
Participated in questionnaire addressed to EU Member States NRL’s to identify the nature of our activities and
expectations of the CRL.
Participated in the questionnaire- The Role of OIE reference Laboratories in providing permanent support for
mandates and objectives of the OIE
Wildlife diseases in UK, case report to Defra & OIE http://www.defra.gov.uk/corporate/vla/science/science-endwildlife
Contributed to Health Protection Agency Zoonoses Report 2007 regarding brucellosis in humans in UK.
Contribution to Environmental Agency report , work on otters
http://publications.environment-agency.gov.uk/pdf/SCHO0307BMKL-e-e.pdf:
agency.gov.uk/pdf/SCHO0307BMKM-e-e.pdf
http://publications.environment-
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2007