AciAr Proceedings 128

Management of classical swine fever and foot-and-mouth disease in Lao Pdr

Management of classical swine fever and foot-and-mouth disease

in Lao PDR

Proceedings of an international workshop held in Vientiane, Lao PDR, 20–21 November 2006

Editors

: J.V. Conlan, S.D. Blacksell, C.J. Morrissy and A. Colling

2008

2

The Australian Centre for International Agricultural Research (ACIAR) was establishedin June 1982 by an Act of the Australian Parliament. Its mandate is to help identifyagricultural problems in developing countries and to commission collaborative researchbetween Australian and developing country researchers in fields where Australia has aspecific research competence.Where trade names are used this constitutes neither endorsement of nor discrimination against any product by the Centre.

ACIAR PROCEEDINGS SERIES

This series of publications includes the full proceedings of researchworkshops or symposia organised or supported by ACIAR. Numbersin this series are distributed internationally to selected individuals andscientific institutions, and are also available from ACIAR’s websiteat <www.aciar.gov.au>.

© Commonwealth of Australia 2008This work is copyright. Apart from any use as permitted under the

Copyright Act 1968

, no part may be reproduced by any process without prior written permission from the Commonwealth. Requests and inquiries concerning reproduction and rights should be addressed to the Commonwealth Copyright Administration, Attorney-General’s Department, Robert Garran Offices, National Circuit, Barton ACT 2600 or posted at <http://www.ag.gov.au/cca>.

Published by the Australian Centre for International Agricultural Research (ACIAR)GPO Box 1571, Canberra ACT 2601, AustraliaTelephone: 61 2 6217 0500; email: <aciar@aciar.gov.au>

Conlan J.V., Blacksell S.D., Morrissy C.J. and Colling A. (eds) 2008. Management of classical swine fever and foot-and-mouth disease in Lao PDR. Proceedings of an international workshop held in Vientiane, Lao PDR, 20–21 November 2006. ACIAR Proceedings No. 128, 98 pp.

ISBN 978 1 921434 98 3 (print)ISBN 978 1 921434 99 0 (online)

Technical editing by Jo Mason, Mason Edit, Adelaide, AustraliaDesign by Clarus Design Pty Ltd, Canberra, AustraliaPrinting by Goanna Print, Canberra, Australia

65

Diagnostic tests for the control of classical swine fever and foot-and-mouth disease

in South-East Asia: An overview

Chris Morrissy1, Lynda Wright1, James Conlan2, Winsome Goff1, Axel Colling1, Jef Hammond1, Michael Johnson1, Stuart Blacksell3 and Peter Daniels1

Abstract

Classical swine fever (CSF) and foot-and-mouth disease (FMD) are two major trans-boundary animaldiseases (TADs) having economic impact on the South-East Asian region. This paper describes the variousdiagnostic tests available for CSF and FMD, the limitations of each and their potential application in a low-technology setting. The need to have complementary field and laboratory operations including suitablesamples and transport methods are discussed, and examples are given. The importance of a qualityassurance system to assess the accuracy and precision of diagnostic results is highlighted.

Introduction

Livestock are highly important in the agriculturallybased economic and social structures of Asia.Endemic and periodically epidemic foot-and-mouthdisease (FMD) has a serious impact on food security(including crop production through its effect ondraught animals), rural income generation, andnational economies by impairing livestock trade.Consequently, the poorest sectors of the community

are the most seriously affected. The progressivecontrol of FMD is both a national and regional pri-ority (Khounsy et al. 2008, in press). FMD is themost contagious disease of mammals and can causesevere economic loss in susceptible cloven-hoofedanimals. While the disease usually does not causehigh levels of mortality, it results in productivitylosses and the lameness it induces severely limits theuses of cattle and buffalo for traction, which is ofmajor importance to the livelihoods of poor farmers.

Classical swine fever (CSF) is known internation-ally as one of the most serious diseases of pigs.Infection may result in mortalities of up to 100% inthe acute form or reproductive failure and increasedsusceptibility to other infections. CSF causes largefinancial losses to both commercial and smallholderpig farmers, contributing to rural poverty. Control ofthe disease is attempted by vaccination. The eco-nomic burden of CSF to the region is difficult toquantify without an accurate diagnostic capability,but there is consensus that it is the most seriousdisease faced by the pig industry.

1 CSIRO Livestock Industries, PO Bag 24, Geelong3220, Victoria, Australia

2 ACIAR Project AH/2003/001, National Animal HealthCentre, Department of Livestock and Fisheries,Ministry of Agriculture and Forestry, Vientiane, LaoPDR

3 Mahidol University–Oxford Tropical MedicineResearch Unit (MORU), Faculty of Tropical Medicine,Mahidol University, 420/6 Rajvithi Rd, Bangkok10400, Thailand; and Centre for Tropical Medicine,Nuffield Department of Clinical Medicine, JohnRadcliffe Hospital, Oxford, UK

66

All farmers and governments in the region spendlarge amounts of money on FMD and CSF vaccinesbut outbreaks still occur, causing the farmers to vac-cinate more frequently. Not all existing laboratoriesin the region have the necessary capability toconfirm the efficacy of FMD and CSF vaccines andto provide an accurate diagnosis. This paper willdiscuss the different options available for the detec-tion of FMD and CSF antigen and antibody that canbe applied to control the diseases. The control oftrans-boundary animal diseases (TADs) such asFMD and CSF can only be achieved by taking aregional approach with countries working together.The application of appropriate diagnostic tests undera quality assurance system to ensure accurate andprecise results, combined with surveillance and adisease investigation program under a well-resourced animal health network, is vital for diseasecontrol.

Animal health network

The diagnosis and control of infectious livestockdisease is an important role of the animal health net-work, comprising both field and laboratory per-sonnel and requiring complementary field andlaboratory operations. The animal health network isactive during periods of disease surveillance andoutbreaks.

During surveillance, laboratory staff conductpost-vaccination testing to both confirm vaccinationsuccess and to determine and monitor disease prev-alence in target populations. The field veterinarianscollect information on vaccination history, whichincludes vaccines used, disease outbreaks and healthstatus of animals. It is important to involve labora-tory diagnosticians, field veterinarians and epidemi-ologists in the planning of surveillance studies. Thiswill ensure that critical parameters, such as suitablesamples, test performance characteristics and accu-rate prevalence estimates, are available for thedesign of sampling frames for surveillance studies.

During a disease outbreak situation, field veteri-narians are charged with the responsibility of col-lecting outbreak information, making a clinicaldiagnosis, collecting samples from suspected cases,and implementing control measures such as disin-fection and quarantine and movement of animals.The laboratory staff support and complement thefield investigation by conducting tests to confirm theclinical diagnosis and isolate the causative agent for

further characterisation, and performing molecularepidemiology studies to establish potential linkswith other outbreaks. In the case of CSF, a labora-tory confirmation is required due to the difficulty ofcorrectly identifying cases based solely on clinicalsigns. For FMD the laboratory confirmation isimportant to establish the serotype responsible forthe outbreak.

Classical swine fever

Clinical diagnosis

It is difficult to accurately and confidently predictthe CSF infection status of a herd based on clinicalfindings alone. The clinical signs and lesions asso-ciated with CSF can vary depending on the virulenceof the virus and, importantly, individual pigs mayshow different signs when infected with the samevirus strain. Clinical diagnosis is further compli-cated by inter-clinician variation. The experience ofthe clinician is very important but, in all cases,samples should be collected and tested in the labo-ratory to confirm or deny a suspicion based on clin-ical findings.

Laboratory diagnosis

The quality of laboratory testing is only as good asthe samples collected and submitted. Suitable sam-ples, which for CSF include spleen, tonsil, lymphnode, whole blood and kidney tissue, will maximisethe chances of making a correct diagnosis. Samplesshould be collected from no fewer than four animalsshowing clinical signs, with samples of approxi-mately 2 g of tissue and 10 mL of blood from eachanimal transported on ice and reaching the labora-tory as soon as possible after collection. A goodhistory of the animals from which the samples werecollected is required, as are details of the outbreakinvestigation or surveillance. This information linksthe diagnostic results with outbreak and controlefforts.

Isolation and specific detection of virus in tissue culture

In-vitro isolation and subsequent detection ofCSF virus is achieved on porcine kidney (PK15)cells or other suitable cells such as primary pigkidney (PK), swine kidney (SK6) or swine testis(ST), and is considered one of the most sensitivediagnostic tests. However, virus isolation (VI)

67

requires specialised facilities for cell culture andhandling of virus, and is expensive to maintain. Ref-erence laboratories require this test for characterisa-tion of virus isolates. CSF virus grows in cell cultureapproximately 18–24 hours after inoculation butsamples must be passaged on cells for three 4-dayperiods before being declared negative. The identi-fication of virus isolates is carried out using specificantiserum- and immuno-techniques on fixed cellcultures, antigen capture (AC)-ELISA orpolymerase chain reaction (PCR) tests, usually48 hours post infection.

Antigen detectionThere are a number of techniques for detection of

antigen, allowing for rapid and cheap detection ofCSF from field samples. In the case of ELISA,testing can be scaled up to process a large number ofsamples in a relatively short period of time. In-houseantigen detection ELISAs (eg AAHL ELISA;Shannon et al. 1993) are commonly used and com-mercial antigen detection ELISA kits are availablefrom many companies, most commonly CEDI Diag-nostics (the Netherlands), IDEXX (USA) and Sym-biotics (France). The antigen detection ELISA forCSF gives a result in 3–5 hours depending on thetest used, with some tests having an overnight incu-bation step. New research in Lao PDR, as part of theACIAR project, has led to the development of arapid antigen detection ELISA test in a tube usingimmunomagnetic beads (IMB) as the solid phase(Conlan 2006; Conlan et al. 2008a). The IMB testcan be used in the field and read by eye with a resultin 60–90 minutes. Immunocytochemistry-basedtests such as the fluorescent antibody test (FAT) andimmunoperoxidase (IPX) staining are also used todetect CSF virus antigen in cell culture and tissuesections, and results can be achieved within 2 hours.

Molecular technologiesWith improvements in PCR technology and

advances in methodologies, the detection of viralRNA as a diagnostic tool has now largely surpassedthe more traditional procedures such as virus isola-tion and FAT. There are a number of conventionaland real-time PCR methods available for the detec-tion of CSF genome. The real-time PCR (Ophuis etal. 2006) methods currently available are rapid andhave high diagnostic sensitivity and specificity.Because the analytical sensitivity of PCR is alsogreater than other tests, viral genome can be

detected in smaller amounts and therefore soonerafter infection, which has important implications forcontrol efforts. Molecular technologies also allowthe investigator to perform genetic characterisationof virus isolates and undertake molecular epidemio-logical studies to identify infection sources and virusevolution. Molecular technologies are, however,expensive and require high-quality samples withintact RNA. When samples are transported atambient tropical temperatures, as is the case in LaoPDR, sample degradation has been shown to be det-rimental to diagnostic performance (Blacksell et al.2004).

Serological detectionDetection of antibodies to CSF virus has limited

scope in diagnosis, particularly if the focus is on theearly detection of virus in a herd or if vaccination isundertaken. Serum antibodies to CSF virus typicallyappear approximately 10–21 days after infection.Serological testing is, however, an important com-ponent of a disease control program to monitor thesuccess of vaccination. Antibody detection is bestachieved by the ‘gold standard’ neutralising peroxi-dase linked assay (NPLA). However, because thistest requires tissue culture, it is time consuming,expensive and not suitable for the rapid screening oflarge numbers of samples. Other methods includein-house ELISAs (Colijn et al. 1999) such as thecomplex trapping blocking (CTB)-ELISA from theAustralian Animal Health Laboratory (AAHL) andELISA kits that can be purchased from commercialsuppliers such as IDEXX and CEDI Diagnostics.Not all diagnostic tests are equally suitable tomonitor sero-conversion after vaccination. Anexample is the AAHL CTB-ELISA that is of limitedvalue to detect post-vaccination antibodies in pigsera because its MAb is specific for the NS3 proteinof the crude antigen extract. These antigens are nor-mally exposed after infection but only in limitedquantities after vaccination. On the other hand, theNPLA and commercial ELISAs, such as the CEDIELISA, are more sensitive for the detection of post-vaccinal antibodies because the CEDI ELISA uses abaculovirus expressed E2 protein subunit and an E2-specific MAb. Under experimental conditions with20 vaccinated pigs, the CEDI ELISA showed asimilar sensitivity to detect post-vaccinal antibodiesas the NPLA, which is considered the gold standard(Conlan et al. in press; Conlan et al. 2008b).

68

Foot-and-mouth disease

Clinical diagnosis

Clinical signs of FMD vary between species. Incattle, onset of FMD is initially characterised bypyrexia, anorexia and shivering, followed bysmacking of the lips, grinding of the teeth, drooling,lameness, and stamping or kicking of the feet. Thesesymptoms are caused by vesicles on buccal andnasal mucous membranes and/or between the clawsand coronary band that will rupture, leaving ero-sions. Recovery generally occurs within 8–15 daysalthough complications can include superinfectionof lesions with bacteria or screwworm infestation,hoof deformation, myocarditis, abortion, death ofyoung animals and permanent loss of weight. Post-mortem lesions on rumen pillars and in the myocar-dium, particularly of young animals (tiger heart),may be evident. In sheep and goats the lesions areless pronounced and foot lesions may go unrecog-nised. Pigs may develop severe foot lesions, partic-ularly when housed on concrete, and there may behigh mortality in piglets. The differential diagnosisis species dependent and includes vesicular stoma-titis, swine vesicular disease and vesicular exan-thema of swine, which are all clinicallyindistinguishable from FMD.

Laboratory diagnosis

Virus isolationAs with CSF, virus isolation is expensive to main-

tain and requires specialised facilities for cell cultureand virus handling. Virus isolation and characterisa-tion is important to compare circulating viruses withvaccine strains (r-value) to maximise vaccine effec-tiveness. FMD virus (FMDV) will grow in a widerange of primary and continuous in-vitro cell cul-tures. The most sensitive cell culture for the isolationof FMDV is primary bovine thyroid (BTY) cells(House and House 1989). Continuous cell lines suchas baby hamster kidney (BHK), lamb kidney (LK)and the pig kidney cell lines IB-RS-2 and MVPK-1are also susceptible to FMDV infection. The sensi-tivity of virus isolation will depend on the quality andtype of cells used as well as the quality of the sample.

Antigen capture ELISAThe antigen capture (AC)-ELISA or serotyping

ELISA is the test of choice for countries endemicwith FMD and is the recommended test for the detec-

tion of FMD antigen (Office International des Epiz-ooties 2004). The FMD AC-ELISA providesdetection of FMD antigen and identification of sero-type in the case of an FMD-positive sample, and wasdeveloped in its current form by Roeder and LeBlanc Smith (1987) and Ferris and Dawson (1988).The FMD AC-ELISA replaced the complement fix-ation test for primary FMD diagnosis and serotypeidentification because of its increased specificity andsensitivity and because it is not affected by pro- oranti-complementary factors in the test sample.Standard reagents for the FMD AC-ELISA are pro-duced at the World Reference Laboratory (WRL) forFMD, Pirbright, United Kingdom. At the RegionalReference Laboratory (RRL), Pak Chong, Thailand,reagents for the detection of serotypes A, Asia 1 andO are routinely produced for use in Asia. Samplequality is important as lesions older than 4–5 dayshave less antigen; however, samples unsuitable forvirus isolation can be tested by ELISA. The ELISAallows high throughput testing of samples and is wellsuited to low-technology settings. Higher throughputcan be achieved with robotics and other equipmentand is mainly used in large laboratories which canafford to purchase and maintain this capability.

Molecular technologiesIn the years since the advent of genetic diagnostic

techniques nearly 2 decades ago, more than 50 dif-ferent nucleic acid hybridisation and various PCRmethodologies have been reported for the diagnosisof FMD. Recently, real-time PCR methods(TaqMan, molecular beacons, Primer-Probe EnergyTransfer system) have been developed for FMDdiagnosis and are now the mainstay for FMD geneticdiagnosis (Reid et al. 2002; Oem et al. 2005). Eval-uation of real-time PCR methods with conventionaldiagnostics (Shaw et al. 2004; Ferris et al. 2006)concluded that PCR was generally more sensitiveand rapid, and is ideal for samples which containlow concentrations of virus. By introducing nucleicacid extraction and pipetting robotics, together withmultichannel real-time PCR machines, diagnosticprocedures have become rapid, robust and auto-mated but may not be best suited to low-technologysettings. Another promising development for devel-oping country laboratories is the one-step, reversetranscription loop-mediated amplification (RT-LAMP) assay, which enables FMD virus to bedetected in under 1 hour in a single tube withoutthermal cycling (Dukes et al. 2006).

69

Serological methodsThe FMD liquid phase blocking (LP)-ELISA was

developed for the detection of FMD antibodiesbecause of the drawbacks of the conventional virusneutralisation tests (VNTs), which included slow-ness of the test (up to 3 days), the use of live virusand cell cultures, and the difficulty in reproducingresults, all which could be countered by the use ofELISA. The FMD LP-ELISA can detect antibodiesagainst all seven FMD serotypes using polyclonalrabbit and guinea pig IgG antibodies to detectresidual FMD antigen following an in-vitro incuba-tion of test serum and FMD antigen (the ‘liquidphase’). Results from the FMD LP-ELISA indicateda high degree of correlation with VNT results forpost-infection and vaccinated animals, and it wassuggested to be a suitable alternative to the VNT(Hamblin et al. 1986a, 1986b, 1987). It was also sug-gested that the FMD LP-ELISA could be used toestimate in-vivo protection to FMD challenge(Hamblin et al. 1986a, 1986b, 1987).

The FMD LP-ELISA is one of the recommendedELISA methods for the detection of FMD antibodies(Office International des Epizooties 2004) and is theprimary test for determining vaccine titres, beingused throughout Asia (Blacksell et al., in press).Recently, the FMD competitive (C)-ELISA hasbeen developed for all seven serotypes of FMD inresponse to the FMD LP-ELISA being less condu-cive to large-scale testing and automation. The FMDC-ELISA was developed using the same reagents asthe FMD LP-ELISA but without the ‘liquid-phase’step, allowing a result in the same day (4–5 hours).The FMD C-ELISA was found to be more robust,sensitive and specific than the FMD LP-ELISA, andwas used in the recent UK FMD outbreak to allowrapid screening of serum samples for FMD anti-bodies.

FMD non-structural protein assays Viral replication in FMD-infected animals

induces an immune response against the non-struc-tural (NS) protein of the FMD. The response againstNS proteins is not serotype specific and indicatesinfection with any of the seven serotypes. Animalswhich are not infected with FMD but vaccinatednormally don’t develop a detectable antibodyresponse against NS protein in the ELISA. Never-theless, repeatedly applied, low-quality vaccines,(e.g. lack of viral inactivation and purification) mayinduce a false positive result in this test. In these

cases the history from the field, e.g. about potentialoutbreaks/infection, identification of vaccine andnumber of vaccinations received, is important forcorrect interpretation of the result.

The use of vaccine for control of FMD has led tothe development of a number of assays for the detec-tion of NS antibodies to discriminate between vac-cinated animals and those that have been infected.AAHL, with the support of IAEA, has developed anin-house FMD NS 3ABC C-ELISA (Morrissy et al.2007). It uses baculovirus expressed 3ABC antigenand a competing antibody, which is produced inchicken. This ELISA has been used and validated inthe region (IAEA TECDOC 2007). There are anumber of commercial ELISA kits (de Bronsvoort etal. 2004) available from CEDI Diagnostics (baculo-virus 3ABC expressed antigen), Bommeli (E-coli3ABC expressed antigen) and UBI (synthetic 3Bantigen), which are the most common NS-ELISAsin use in the region. The CEDI Diagnostics kit andthe AAHL kit are both competitive ELISAs that canbe used for all species, whereas the other kits areindirect ELISAs with separate kits for ruminants andpigs. The CEDI Diagnostics kit has been found to bethe most sensitive and specific kit of those used inthe region (Brocchi et al. 2006). Comparisonsbetween the kits from CEDI and AAHL have shownthat both ELISAs have similar performance charac-teristics when applied in the region.

Quality assurance and quality control (QA/QC)

‘Quality is fitness for the intended purpose’. Qualityassurance (QA) is a system designed to assure testfacility management of compliance with a qualitystandard, e.g. AS ISO 17025-2005 ‘General require-ments for the competence of testing and calibrationlaboratories’. Quality control (QC) is the technicalrealisation of the QA concept, e.g. calibration, assayvalidation, precision and accuracy of test results.QA and QC principles are crucial requirements tocomply with quality standards such as ISO 17025-2005 or the OIE’s ‘Quality standard and guidelinesfor veterinary laboratories: Infectious diseases’.

The key components of QA are:• paperwork/documentation of all tests into

standard protocols• validation data for diagnostic tests being used in

the laboratory

70

• staff training and accreditation • internal quality control (IQC)—positive and

negative controls included in each test run• analysis and charting to document results from

IQC controls used in each test• external quality assurance—successful partici-

pation in proficiency test rounds• documentation on all sample collection, storage

and transport from the field, and storage andhandling in the laboratory

• calibration of equipment and calibration records• laboratory accreditation to a standard, e.g. ISO

17025-2005.Quality control of diagnostic tests is achieved

through a combination of IQC and external qualityassurance (EQA). Repeatability and reproducibilityare measurements of precision and results are of par-ticular value to monitor the validity of test results(De Clercq et al. 2008).

IQC is useful to measure the repeatability of testresults in a laboratory. Ideally, internal controlsshould be included as replicates in each test run andshould cover at least the critical range of test resultsto be expected, e.g. strong positive control (C++);weak positive control, which is slightly above thecut-off (C+); and negative control. Analysis of IQCswill give information about intra- and inter-assayvariation, intra- and inter-operator variation, day-to-day variation etc. Critical parameters are basic sta-tistics such as mean values, standard deviation,coefficient of variation, range, and upper and lowercontrol limits. Results can be charted and recordedas Levey-Jennings charts. This approach helps toidentify trends in assay performance and is useful toprompt preventive corrective actions or trouble-shooting. IQC data can also be useful to assessmeasurement of uncertainty, e.g. continued meas-urement of replicates of an internal positive controlclose to the cut-off (see <http://www.scahls.org.au/policyguidelines/Worked_MU_examples.doc>).

EQA or proficiency testing (PT) measures thereproducibility of a test and its performance in dif-ferent laboratories. It helps to standardise test resultsfor the same test in different laboratories (inter-labora-tory comparison, ring test or external quality assur-ance) or to harmonise test results from different tests indifferent laboratories (proficiency test round). Suc-cessful and regular participation approximately twicea year in EQA programs is an essential component ofISO 17025-2005 or OIE quality standard require-ments, and therefore a pre-condition for accreditation.

Equipment calibration and maintenance isanother important part of QA because it helps toensure that tests are giving correct results. It isimportant that laboratories have a budget to allowthem to maintain and calibrate their equipment. Insummary, QA and QC are crucial elements in a lab-oratory’s quality system and need to be well estab-lished to achieve accreditation to internationallyaccepted standards.

Discussion

Effective diagnosis and control of livestock diseasesrequires a strong animal health network where labora-tory staff, field veterinarians and epidemiologistswork together. Laboratories contributing to the diag-nostic network must be able to carry out diagnosiswith OIE recommended or alternative tests within arecognised QA system. OIE reference laboratoriesplay an important role in monitoring the disease situa-tion in a country and ensuring that continued, updatedand accurate information is forwarded to OIE. This isespecially important with TADs, zoonotic, and newand emerging diseases because of their global threat.

The laboratory network in a country is made up oflaboratories at different levels of standard and capa-bility, from the national laboratory down to theprovince and district levels. The diagnostic testsused in these laboratories will differ according totheir respective capabilities (Tables 1 and 2).

The national laboratory may have the full range ofdiagnostic tests, which includes virus isolation and amolecular capability for PCR and sequencing,whereas a provincial or district laboratory will onlyhave low-cost technology. Tests such as ELISAs arethe most routinely used for CSF and FMD antibodyand antigen detection. ELISAs are cheaper to run thanvirus isolation and PCR but reagents and equipmentare still expensive for laboratories in poorer countriesor at the district level. The development of cheaper orlow-technology diagnostic tests such as the IMB-ELISA for CSF is important to allow rapid diagnosisclose to the disease outbreak, e.g. in a district labora-tory. The IMB-ELISA does not require any expensiveequipment and can be easily quality assured.

For CSF serology the ELISA is the test of choicefor sero-surveillance and post-vaccination testing.The VNT gives greater sensitivity and is used tosupport or confirm ELISA results. Normally it isavailable either at the national laboratory or a refer-ence laboratory. The VNT test is still the test of

71

Tab

le 1

. C

ompa

riso

ns a

mon

g cl

assi

cal s

win

e fe

ver

diag

nost

ic te

sts

Typ

e of

test

DSn

DSp

Spee

dC

ost

Qua

lity

of

sam

ple

requ

ired

Deg

ree

of

prof

icie

ncy

requ

ired

Hig

h sa

mpl

e th

roug

hput

App

licab

ility

to

ref

eren

ce

labo

rato

ry

App

licab

ility

in

a lo

w-

tech

nolo

gy

setti

ng

Vir

us is

olat

ion

••••

•••

•••

•••

•••

••••

•••

••

••••

••

Mol

ecul

ar te

chno

logi

es••

•••

••••

•••

••••

•••

••••

•••

•••

••••

••••

•••

•

AC

-EL

ISA

•••

••••

••••

•••

•••

••••

•••

•••

•••

IMB

-EL

ISA

•••

••••

••••

••

•••

•••

•••

•••

•••

Imm

uno-

cyto

chem

istr

y••

••••

••••

••••

••••

••••

••••

•••

••••

Sero

logy

••••

••••

••••

••••

•••

•••

••••

•(N

PLA

and

E

LIS

A)

••••

•(E

LIS

A)

DSn

= d

iagn

ostic

sen

sitiv

ity; D

Sp =

dia

gnos

tic s

peci

fici

ty

Tab

le 2

. C

ompa

riso

ns a

mon

g fo

ot-a

nd-m

outh

dis

ease

dia

gnos

tic te

sts

Typ

e of

test

DSn

DSp

Spee

dC

ost

Qua

lity

of

sam

ple

requ

ired

Deg

ree

of

prof

icie

ncy

requ

ired

Hig

h sa

mpl

e th

roug

hput

App

licab

ility

to

ref

eren

ce

labo

rato

ry

App

licab

ility

in

a lo

w-

tech

nolo

gy

setti

ng

Vir

us is

olat

ion

••••

(BT

Y)

••••

•(B

TY

)••

••••

•••

•••

••••

••••

•••

•

Mol

ecul

ar te

chno

logi

es••

•••

••••

•••

••••

•••

••••

•••

•••

••••

•••

•••

••

AC

-EL

ISA

•••

••••

•••

•••

•••

•••

•••

••••

•••

••••

•••

LPB

-EL

ISA

••••

••••

••••

•••

•••

•••

••••

••••

••••

•

BT

Y =

bov

ine

thyr

oid

cells

(B

TY

is m

ost s

ensi

tive

cell

line;

oth

er c

ell l

ines

are

less

sen

sitiv

e)D

Sn =

dia

gnos

tic s

ensi

tivity

; DSp

= d

iagn

ostic

spe

cifi

city

72

choice when studying maternal antibody levels inpiglets to determine the best time for vaccination andvaccine protocols. For detection of CSF, an ELISAis recommended, especially where large numbers ofsamples are being tested. The CSF PCR for detec-tion of genome is recommended where the labora-tory has the capability in place and is testing smallnumbers of samples. Virus isolation is important forfurther characterisation and is best carried out in thenational laboratory or a reference laboratory.

For FMD serology the LP-ELISA is the test ofchoice in a country where FMD is endemic, as it isstill the only test validated for post-vaccinationtesting. The C-ELISA is used in FMD-free coun-tries, and can be used to screen sera first as it hasgreater sensitivity and specificity and allows greaterthroughput. The NS-ELISA is used with the struc-tural LP-ELISA or the C-ELISA to distinguishinfected animals from vaccinated. The NS-ELISAdoes not identify carrier animals, but rather animalsthat have been infected with FMDV in the past. TheNS-ELISA can be used to indicate disease preva-lence, or when a country is declaring freedom fromFMD, or in animal trading to indicate that animalshave not been exposed to FMDV. The AC-ELISA isused for detection of FMD antigen and is the onlytest able to rapidly determine the serotype of anFMD outbreak. PCR is important as a confirmationfor FMD genome and in further characterisation ofFMDV by sequencing. Virus isolation is importantin producing high-titred stocks of FMDV for char-acterisation or for growth of samples with low virustitre. Virus isolation is used in national or referencelaboratories due to the high cost of maintainingtissue culture.

The quality of samples submitted to the laboratoryis important in achieving precise and accurateresults and involves:• maintaining a cold chain• collection of appropriate samples for diagnosis • collection in the appropriate sample collection

buffer (i.e. phosphate/glycerol for virus isolationand ELISA).Training of laboratory staff in the different diag-

nostic tests for FMD and CSF is an important part ofAAHL’s overseas projects. Training includesaspects of test validation and application of internaland external quality control and assurance principlesto monitor assay reliability. Quality results enableepidemiologists and policymakers to makeinformed decisions about animal health policies.

References

Blacksell S.D., Khounsy S., Conlan J.V., Gleeson L.J.,Colling A. and Westbury H.A Foot-and-mouth diseasein the Lao PDR: II. Sero-prevalence estimates usingstructured surveillance and abattoir surveys. RevueScientifique et Technique (International Office ofEpizootics), in press.

Blacksell S.D., Khounsy S. and Westbury H.A. 2004. Theeffect of sample degradation and RNA stabilization onclassical swine fever virus RT-PCR and ELISA methods.Journal of Virological Methods, 118(1), 33–37.

Brocchi E., Bergmann I.E., Dekker A., Paton D.J., SamminD.J., Greiner M., Grazioli S., De Simone F., Yadin H.,Haas B., Bulut N., Malirat V., Neitzert E., Goris N.,Parida S., Sørensen K. and De Clercq K. 2006.Comparative evaluation of six ELISAs for the detectionof antibodies to the non-structural proteins of foot-and-mouth disease virus. Vaccine 24, 6966–6979.

Colijn E.O., Bloemraad M. and Wensvoort G. 1997. Animproved ELISA for the detection of serum antibodiesdirected against classical swine fever virus. VeterinaryMicrobiology 59, 15–25.

Conlan J. 2006. Improved diagnostics and management ofclassical swine fever virus in Laos. MSc thesis, School ofVeterinary Medicine, University of Melbourne.

Conlan J., Khounsy S., Blacksell S.D., Morrissy C.J.,Wilks C.R. and Gleeson L.J. Development andevaluation of a rapid immunomagnetic bead assay forthe detection of classical swine fever virus antigen.Tropical Animal Health and Production, in press.

Conlan J., Khounsy S., Phruaravanh M., Soukvilai V.,Morrissy C., Colling A., Blacksell S., Wilks C. andGleeson L. 2008a. Application of immunomagneticbead technology for improved diagnosis of classicalswine fever virus. These proceedings.

Conlan J., Vitesnik T., Khounsy S., Wilks C. and GleesonL. 2008b. Classical swine fever virus vaccine stability inLao PDR. These proceedings.

de Bronsvoort B.M.C., Sørensen K.J., Anderson J.,Corteyn A., Tanya V.N., Kitching R.P. and Morgan K.L.2004. Comparison of two 3ABC enzyme-linkedimmunosorbent assays for diagnosis of multiple-serotype foot-and-mouth disease in a cattle population inan area of endemicity. Journal of Clinical Microbiology42(5), 2108–2114.

De Clercq K., Goris N., Barnett P.V. and MacKay D.K.2008. The importance of quality assurance / qualitycontrol of diagnostics to increase the confidence inglobal foot-and-mouth disease control. Transboundaryand Emerging Diseases 55, 35–45.

Dukes J.P., King D.P. and Alexandersen S. 2006. Novelreverse transcription loop-mediated isothermalamplification for rapid detection of foot-and-mouthdisease virus. Archives of Virology 151, 1093–1106.

73

Ferris N.P. and Dawson M. 1988. Routine application ofenzyme-linked immunosorbent assay in comparisonwith complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. VeterinaryMicrobiology 16, 201–209.

Ferris N.P., King D.P. and Reid S.M. 2006. Comparisons oforiginal laboratory results and retrospective analysis byreal-time reverse transcriptase-PCR of virologicalsamples collected from confirmed cases of foot-and-mouth disease in the UK in 2001. The Veterinary Record159, 373–378.

Hamblin C., Barnett I.T. and Crowther J.R. 1986a. A newenzyme-linked immunosorbent assay (ELISA) for thedetection of antibodies against foot-and-mouth diseasevirus. II, Application. Journal of ImmunologicalMethods, 93, 123–129.

Hamblin C., Barnett I.T.R. and Hedger R.S. 1986b. A newenzyme-linked immunosorbent assay (ELISA) for thedetection of antibodies against foot-and-mouth disease.I. Development and method of ELISA. Journal ofImmunological Methods 93, 115–121.

Hamblin C., Kitching R.P., Donaldson A.I., Crowther J.R.and Barnett I.T.R. 1987. Enzyme-linkedimmunosorbent assay (ELISA) for the detection ofantibodies against foot-and-mouth disease virus. III.Evaluation of antibodies after infection and vaccination.Epidemiology and Infection 99, 733–744.

House C. and House J.A. 1989. Evaluation of techniques todemonstrate foot-and-mouth disease virus in bovinetongue epithelium: comparison of the sensitivity ofcattle, mice, primary cell cultures, cryopreserved cellcultures and established cell lines. VeterinaryMicrobiology 20, 99–109.

IAEA TECDOC 1546. 2007. The use of non-structuralproteins of foot-and-mouth disease virus (FMDV) todifferentiate between vaccinated and infected animals.At: <http://www-naweb.iaea.org/nafa/aph/public/te_1546_web.pdf>.

Khounsy S., Conlan J.V., Gleeson L.J., Westbury H.A.,Colling A., Paton D.J., Knowles N.J., Ferris N.P. andBlacksell S.D. Foot-and-mouth disease in the Lao PDR:

I, A review of recent outbreaks and lessons from controlprograms. Revue Scientifique et Technique(International office of Epizootics), in press.

Morrissy C.J., McEachern J., Colling A., Wright L., LongN.T., Goff W., Hammond J., Johnson M., Crowther J.R.,Hoa D.M. and Daniels P. 2007. International validation ofa new 3 ABC competitive ELISA for FMD serology.Proceedings of World Association of VeterinaryLaboratory Diagnosticians 13th International Symposium,Melbourne, Australia, 12–14 November 2007.

Oem J.K., Kye S.J. and Lee K.N. 2005. Development of aLightcycler-based reverse transcription polymerasechain reaction for the detection of foot-and-mouthdisease virus. Journal of Veterinary Science, 6, 207–212.

Office International des Epizooties. 2004. Foot and mouthdisease. In: ‘Manual diagnostic tests and vaccines forterrestrial animals (mammals, birds and bees)’. OfficeInternational des Epizooties.

Ophuis R.J.A.O., Morrissy C.J. and Boyle D.B. 2006,Detection and quantitative pathogenesis study ofclassical swine fever virus using a real time RT-PCRassay. Journal of Virological Methods 131(1), 78–85.

Reid S.M., Ferris N.P. and Hutchings G.H. 2002. Detectionof all seven serotypes of foot-and-mouth disease virus byreal-time, fluorogenic reverse transcription polymerasechain reaction assay. Journal of Virological Methods105, 67–80.

Roeder P.L. and Le Blanc Smith P.M. 1987. Detection andtyping of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid andreliable technique for primary diagnosis. Research inVeterinary Science 43, 225–232.

Shannon A.D., Morrissy C.J., Mackintosh S.G. andWestbury H.A. 1993. Detection of hog cholera virusantigens in experimentally-infected pigs using anantigen-capture ELISA. Veterinary Microbiology 34,233–248.

Shaw A.E., Reid S.M. and King D.P. 2004. Enhancedlaboratory diagnosis of foot and mouth disease by real-time polymerase chain reaction. Revue Scientifique etTechnique 23, 1003–1009.