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