b. abortus as viewed by electron microscopy. abortus as viewed by electron microscopy cells are...
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B. abortus as viewed by electron
microscopy
Cells are approximately 0.5-0.7 µm in
diameter and 0.6-1.5 µm in length
Brucella
Establishment of a multi-sectoral strategy for the control
of brucellosis in the main peri-urban dairy production
zones of West and Central Africa (BBSRC-ZELS)
By
John McGiven
(john.mcgiven@apha.gsi.gov.uk)
Serology Workshop
Presentation overview
• Who I am & why am I here?
• Aims of the workshop
• Brucella
• Serodiagnosis of brucellosis (overview)
• Standardisation of serodiagnosis
• ZELS serology: seroprevelence study
• Methods: Milk indirect ELISA
Competitive ELISA (serology)
Rose Bengal Test
• Testing process
• Proficiency testing
• ZELS serology: DIVA development &
mobile diagnosis
John McGiven
• Animal & Plant Health Agency (Weybridge)
• National & OIE (World Organisation for Animal Health)
Reference Laboratory for Brucellosis
• FAO/WHO Collaborating Laboratory for Brucellosis
Routine serodiagnosis (trade/surveillance/AI)
Bacterial culture
Applied Research & Development
Molecular (DNA) epidemiology & detection
Imunodiagnostic assay development
• Provide training in conventional serology to be applied for
ZELS seroprevelence study
• Enable you to effectively apply these methods in your
laboratories
• Provide direct access to diagnostics provider now and for
the future
• To create a personal network of colleagues & make friends
• To enable the successful conclusion of the seroprevalence
study
Serology workshop: Aims
Brucellosis
• Brucellosis is caused by the
intracellular pathogen Brucella
• They are Gram-negative coccobacillary
rods
• The Genus includes six classical
species (B. abortus, B. melitensis, B.
suis, B. canis, B. ovis & B. neotomae),
• B. abortus, B. melitensis & B. suis are
smooth (possess O-polysaccharide
[OPS]) & cause the most disease in
livestock & humans
• B. ovis & B. canis naturally rough –
have lost OPS
Host Range
• Members of the genus Brucella may be found across a very wide host
range (horses, wild boar, rodents, hares, camels, bison, elk, reindeer,
alpaca, chamois, seals, dolphins, proposes, baboons, bullfrogs, foxes….)
• However, different spp. do display tropism for primary hosts & disease
may be different or absent in secondary hosts – but infection may still be
of epidemiological significance
• Brucella are highly conserved & basis for host preference not understood
• B. abortus primarily infect large ruminants
• B. melitensis primarily infect small ruminants
• B. suis biovars 1, 2, or 3 primarily infect swine
• B. ovis primarily infect sheep
• B. canis primarily infected dogs
• B. ceti primarily infect dolphin or porpoise (dependent on clade)
• B. pinnipedialis primarily infect seals
Serology – why is it so important?
• Silent disease (animals): lack of clinical signs
• Cultural diagnosis is difficult, dangerous & expensive
• PCR from blood or tissue samples is currently unreliable
• Ease of access to serum & comparatively low cost of serology
• Variety of serological test platforms to suit laboratory conditions & needs
• Amenable to high-throughput testing
• Serology has good diagnostic sensitivity due to strong induction of Abs against the humorally immunodominant Brucella OPS
• All routine (approved OIE) serodiagnostic assays are dependent upon the inclusion of significant quantities of OPS within the diagnostic antigen
• Includes: whole cell (e.g. RBT/SAT/CFT) sLPS (i/c ELISA) OPS (FPA)
• These assays are the mainstay of eradication and surveillance schemes for achieving and maintaining OIE officially brucellosis free (OBF) status
• Whole cell antigen tests• Serum agglutination / Wright test (1897)
• Complement Fixation Test (1963)
• Rose Bengal Test (1967) & BPAT (1984)
• Eradication and control of brucellosis is possible with these tests (e.g. GB 1985)
Classical Serology
Objective & quantitative
– sLPS iELISA (1976)
– sLPS cELISA (1989)
– OPS FPA (1996)
• All these tests are successful due to the
immunodominance of the OPS antigen
• And their standardisation to ensure maintenance
of optimal diagnostic efficacy
Contemporary Serology
OIE Reference Laboratory Role
• Internationally recognised procedures for disease diagnosis and
vaccination
• Methods standardised & defined
• Used for International Trade testing
• Surveillance to demonstrate
disease freedom
• Reviewed and updated by international
experts in pertinent diseases
• OIE Labs to assist with training &
dissemination of information regarding
best practice
• APHA has more OIE Ref Labs than
any other institution
Standardisation of serodiagnostic tests
• OIE Manual, Chapter 2.4.3. Bovine brucellosis (2. Serology) http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.03_BOVINE_
BRUCELL.pdf
• Specifically describes standardised protocols for antigen production….
• And assay procedures (e.g. for RBT, BPAT, CFT, iELISA, cELISA, FPA)
• Also describes the use of OIE International Standard Sera to ensure that
each assay meets minimum standards of diagnostic efficacy
• The interpretation of assay results based upon existing validation and
historical data is only appropriate if the OIE standards have been met
• The RBT antigen is standardised so that (when the correct method is
applied it will be positive with a 1/45 dilution of the OIEISS and negative
with a 1/55 dilution
• The ELISA kits are standardised so that (when the correct method is
applied and the internal control conditions are met) the OIE ELISA
positive and negative standards are positive and negative when tested
• Good quality assays give good quality results !
c1 c2
c4c6
c1
c1
c1
c2
c2
c2
Reducing end
Non-reducing end
OPS: the Immunodominant humoral antigen
The OPS from smooth Brucella (B. abortus, B. melitensis & B. suis)
is an unbranched homopolymer of 4-6-dideoxy-4-formamido-
mannopyranosyl (D-Rha4NFo), average length ~ 44 units
Image adapted from Haag et al 2010
Indirect (i) ELISA
• Pre-coated antigen
ELISA plates
• Add serum/milk,
incubate
• Add anti-antibody
HRP conjugate,
incubate
• Add HRP substrate,
incubate
• Stop reaction &
read absorbance
• Wash
• Wash
Polystyrene ELISA plate surface
Brucella sLPS
HRP conjugated rabbit
anti-bovine IgG1
ABTS
(Absorbance
max at 405 nm)
Brucella milk iELISA (for detection
of Abs against Brucella sLPS)
• Add 50 µl of diluting buffer to each well of sLPS pre-coated ELISA
plate
• Add 50 µl of test milk samples to wells of ELISA plate (columns 1-10)
• Add 50 µl of control milk samples to wells of ELISA plate (columns 11-
12) – well H12 has no milk added and is the ‘blank’
• Incubate for 30 mins at room-temp on rotary shaker & wash plate 5x
• Add 100 µl of anti-bovine HRP conjugate (pre-diluted to working
strength) to all ELISA wells
• Incubate for 30 mins at room-temp on rotary shaker & wash plate 5x
• Add 100 µl of working strength ABTS substrate and H2O2 donor to all
ELISA wells and incubate at room temp on bench for ~ 15 mins
• Add sodium azide stopper and read ELISA well absorbance values
(Optical Density, OD) at 405 nm
Brucella milk iELISA (for detection
of Abs against Brucella sLPS)
• Milk iELISA sample addition
1. Test samples:
columns 1-10,
1 sample per well
2. Positive control:
A11 – H11
3. Medium positive
control: A12 - D12
4. Negative control:
E12 – G12
5. Blank: H12
Brucella milk iELISA: Data Analysis
• Perform Quality Control Calculations on the control values to ensure
that the data is of sufficient quality for further use
OD of the blank well is < 0.1
Subtract the blank OD from all other OD values before further
calculations
The mean OD of the negative control wells is < 0.1
The mean OD of the positive control wells is > 0.7
The Binding Ratio is > 10 (mean of positive controls / mean of
negative controls)
The mean of the medium controls is between 10% and 30% of the
mean OD of the positive control
• If all of these criteria pass then the test results can be used
• Results for test samples calculated as:
(test sample OD / mean OD of positive controls) x 100 (to convert to %)
• Results > (or equal to) 10.0% are considered positive
Brucella milk iELISA: cream layer
• In some cases, cream from the sample can cause a false positive
reaction due to its ‘stickiness’.
• Care should be taken to avoid adding excess cream to the ELISA plate
with the milk sample
• Cream can be avoided by penetrating the cream layer with the pipette
tip prior to aspiration of the milk & wiping the tip with a clean tissue
before dispensing into the ELISA plate
• Depending on the milk recepticle it may be possible to tip the container
and retain some milk within the lid that can be aspirated
• If there is concern that excess cream may have contributed to a
positive test result, the sample should be retested with extra care
taken to avoid the cream and this retest result used (although the data
from both tests should be retained)
Negative Positive
Competitive (c) ELISA
• Simultaneous incubation of serum and anti-Brucella
sLPS MAb (HRP conjugated)
• No serum Abs to sLPS →
uninhibited MAb binding &
colour development
HRP
conjugated
Mouse anti-
Brucella sLPS
IgG
Serum anti-
Brucella sLPS
antibody
• Specific serum Abs present
→ competition between Abs
and MAb for sLPS binding
Brucella sLPS
Brucella cELISA (for detection of
Abs against Brucella sLPS)
• Add 20 µl of test serum to wells of sLPS pre-coated ELISA plate
(colums 1 – 10)
• Add 20 µl of positive control serum to wells F11 – H12
• Add 20 µl of negative control serum to wells A11 – C12
• Add 100 µl of HRP-conjugated MAb (in diluting buffer) to all wells
• Shake plate vigorously for 2 mins to mix serurm and conjugate
• Incubate for 30 mins at room-temp on rotary shaker & wash plate 5x
• Add 100 µl of OPS substrate and H2O2 donor (in water) to to all ELISA
wells and incubate at room temp on bench for ~ 15 mins
• Add 100 µl of citric acid stopper to all wells and read ELISA well
absorbance values (Optical Density, OD) at 450 nm
Brucella cELISA (for detection of
Abs against Brucella sLPS)
• cELISA sample addition
1. Test samples:
columns 1-10,
1 sample per well
2. Positive control:
F11 – H1211
3. Negative control:
A11 - C12
4. Conjugate control:
D11 – E12
Brucella cELISA: Data Analysis
• Perform Quality Control Calculations on the control values to ensure
that the data is of sufficient quality for further use
The mean OD of the 6 negative control wells is > 0.700
The mean OD of the 6 positive control wells is < 0.100
The mean OD of the 4 conjugate control wells is > 0.700
The Binding Ratio is > 10 (mean of 6 negative controls / mean of 6
positive controls)
• If all of these criteria pass then the test results can be used
• Results for test samples calculated as:
(test sample OD / mean OD of conjugate controls) x 100 (to convert to %)
• Results < (or equal to) 60.0% are considered positive
• High quality ELISAs are worthless without data traceability
• Essential that sample IDs are correctly matched to test results
• Keep all data: raw data (raw plate ODs and control value ODs) as well as processed data (percentage of control)
• The raw data may prove essential in resolving any outliers that may become apparent later in the study
• The raw data may also prove essential if assay pos/neg cut-offs need to be adjusted to suit local conditions
• Keep it all !
ELISA data traceability
• RBT antigen must be mixed before use to evenly suspend cells
• RBT antigen must be brought to room temperature before use (remove required amount from bottle – do not repeatedly warm and chill main stock)
• Add 30 µl of serum to a white tile & 30 µl of antigen
• Positive and negative control sera must be used each time test is performed
• Mix together using a clean utensil (e.g. pipette tip)
• Agitate on rocker for 4 mins
• Then read agglutination
• Results are +ve or -ve
Rose Bengal Test
Negative Positive
• Practical workshop this week: 1 day (am & pm session) per group
• Milk indirect ELISA, Serum competitive ELISA & RBT
• Everyone to have a go and please ask questions
• These methods to be implemented in your labs
• The kits will be sent by APHA (me)
• We will also send you a blinded proficiency testing serum panel to evaluate your capability to perform each test effectively
• We require you to send back your test results for the serum panel
• I will send you back a collated version of the results from all the participants
• The results will be coded so that only you (and the project co-ordinators) will be able to identify your results
Training & Implementation
Next Phase: DIVA Assays
The most protective Brucella vaccines contain OPS (B. abortus S19
& B. melitensis Rev1)
OPS is a key ingredient in optimal vaccine formulations
Significant problem with such vaccines is induction of anti-OPS
antibodies and interference with serology
Therefore very difficult to run concurrent vaccination and test and
slaughter disease control/eradication schemes
Also makes it more difficult to estimate efficacy of vaccination
programmes as serology is removed from the tool box
OIE will not allow declaration of OBF status if vaccination has been
used within 3 years
Cessation of vaccination to switch to test and slaughter is therefore
a decision that caries considerable risk due to re-emergence of
disease
DIVA assay would be highly desirable
Eliminate OPS from vaccine & use OPS based antigens as
diagnostics
Rough (or subunit) vaccines not as protective (e.g. B. abortus RB51)
Conventional DIVA rationale
Find alternative diagnostic antigen to OPS and knock this ‘marker’
out of a vaccine candidate to create DIVA vaccine & assay
But no effective diagnostic alternatives to LPS have been identified
“The attempts to find a ‘magic’ protein antigen for differentiating smooth
Brucella-infected animals from vaccinated individuals have been finally
relegated as experimental relics, mainly because the sensitivity and
specificity of protein based assays did not rival those developed with LPS”
(Chaves-Olarte et al., 2012)
Protein antigens do not appear to be part of the solution
Test mobility
Development of an Lateral Flow Device for the
detection of anti-Brucella antibodies in serum
Simple
Portable
Robust
Sensitive
Specific
Durable
Standardised
Novel (potentially)
Lateral Flow Devices for ‘pen-side detection’
Sample
Indirect formats e.g.
FMD virus (Ferris et al 2009)
Swine Vesicular Disease virus
(Ferris et al 2009)
Anti-Anaplasma marginale
antibody (Nielsen et al 2009)
Anti-Brucella antibody detection
(Abdoel et al 2007)
Antibovine
Brucella sLPS
anti-sLPS Serum antibody
Acknowledgements
Defra (UK Gov)
APHA Brucella workgroup
Dr Adrian Whatmore
Laurence Howells
Lucy Duncombe
Andrew Taylor
Lorraine Perrett
The Royal Veterinary College
Professor Javier Guitian
Laura Craighead
EISMV
Professor Kone
Professor Ayih-Akakpo
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