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Airborne transmission of foot-and-mouth disease in pigs:Evaluation and optimisation of instrumentation and techniques

Claudia M.F. Amaral Doel a, * ,1 , John Gloster a,b,1 , Jean-Francois Valarcher c

a Institute for Animal Health, Pirbright Laboratory, Woking, Surrey GU24 0NF, UK b Met Office, FitzRoy Road, Exeter EX1 3PB, UK

c IVI Animal Health, La rkbacken, 740 20 Va nge, Uppsala, Sweden

Accepted 6 September 2007

Abstract

Foot-and-mouth disease (FMD) can be transmitted in a variety of ways, one of which is through virus exhaled into the air by infectedlivestock. It is clear that where there is close contact there will be a range of possible mechanisms for the transmission of disease fromanimal to animal, including the airborne route if simple barriers between livestock exist. In transmission of FMD over longer distances,airborne transmission represents a signicant challenge to the veterinary services in that the mechanism is essentially uncontrollable if theprimary source of the disease is not contained. In the event of an epidemic of FMD, such as the one experienced in the United Kingdomin 2001, it is important for disease control purposes to understand the contribution made to the overall spread of disease by aerosolisedvirus. This assessment is based on a combination of measurements made in the laboratory and through clinical observations in the eld.To date, laboratory measurements have used a number of instruments that were not specically designed for working with FMD virus orwhose performance have not been fully compared and documented. This paper compares four samplers and describes the method bywhich samples are processed. Overall it is concluded that there is no optimum air sampling instrument which could be successfullyemployed for all situations but the work provides guidance to those wishing to make measurements in the future and establishes a base-line against which any new samplers can be compared. 2007 Elsevier Ltd. All rights reserved.

Keywords: Foot-and-mouth disease; Airborne; Air samplers; Spread

Introduction

Pigs, cattle, sheep and other ruminants infected withfoot-and-mouth disease virus (FMDV) emit aerosol parti-cles into the atmosphere. These can then be inhaled or

ingested by other animals which subsequently becomeinfected. A comprehensive review of the pathogenesis of the disease is given by Alexandersen et al. (2003) . Althoughairborne infection of FMD was rst hypothesised in theearly 1900s (Penberthy, 1901; Bang, 1912 ) and there havebeen numerous results from laboratory experiments, noinstrument capable of specically measuring FMDV

has ever been specically designed and made generallyavailable.

Measurements have been taken with a range of instru-ments, mainly developed in the 1960s, and the samples ana-lysed using a number of laboratory techniques. The

samplers used have included the Cyclone, Litton, Porton,May and the SKC BioSampler ( Errington and Powell,1969; Donaldson et al., 1982; May and Harper, 1957;May, 1966 ). Whilst each instrument and sample analysistechnique has been described in the literature, no compre-hensive intercomparison has been made, nor have theinstruments been optimised for FMDV sampling and theresults documented. This paper addresses these shortcom-ings by comparing the Cyclone, Porton, May and SKCBioSampler under controlled and realistic laboratory con-ditions. The objective was to offer guidance to others

1090-0233/$ - see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tvjl.2007.09.010

* Corresponding author. Tel.: +44 1483 232 441; fax: +44 1483 232 448.E-mail address: [email protected] (C.M.F. Amaral Doel).

1 Joint rst authors.

www.elsevier.com/locate/tvjl

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The Veterinary Journal 179 (2009) 219224

The Veterinary J
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involved in sampling from FMD infected animals and toprovide a baseline by which new instruments can be com-pared. For mathematical modelling purposes, our nalresults took into account the duration of the air samplingperiod, the volume of sample collected and the ow rateof the air during sampling.

Materials and methods

Previous studies have shown that pigs, the most prolic of virusemitters, when infected with FMD can excrete virus in their breath for aperiod up to 45 days and maximum excretion occurs during the veryearly stages of acute disease ( Alexandersen et al., 2003; Gloster et al.,2006). Consequently we decided to infect a group of pigs with FMDV,make a detailed clinical assessment of the animals on a daily basis andschedule aerosol measurements from 14 days post-infection (d.p.i.).

Animal experimentation was carried out in accordance with the UKAnimals (Scientic Procedures) Act 1986 and associated guidelines.

Animals

Six Large White cross Landrace pigs, 2030 kg in weight, were used inthe experiment. They were housed in a biosecure isolation building andkept in a loosebox of 3.6 m 3.3 m 3.0 m for the duration of theexperiment.

Each pig was inoculated intradermally in the heel bulb of the left rearfoot with approximately 10 5.0 TCID 50 (BTY) in a dose volume of 0.5 mL(0 d.p.i.). The pigs were examined daily for clinical evidence of FMD andscored using a modied version of the clinical scoring framework pub-lished by Quan et al. (2004) with a range of score from 017. A numericalscore is given when each animal is examined according to its temperature,the presence of clinical signs on each of the feet, snout, mouth, tongue,teats, and noting whether the pig is lame and/or there are any othercharacteristics (off food/water, behaviour changes, nasal discharge, sec-ondary infection). Serum samples of all six pigs were taken on a daily

basis, starting just before needle inoculation of virus (04 d.p.i.).

Virus

FMDV strain O UKG 34/01 was isolated from a pig at Cheale MeatsAbattoir, Brentwood, Essex, UK, on the 20th February 2001. This waspassaged once in pigs and the stock virus produced from foot lesion epi-thelium had a nal titre of 10 7.0 TCID 50 /mL in primary cultures of bovinethyroid (BTY) cells (Snowdon, 1966). Tissue culture infectious dose(TCID 50 ) is the dose of agent (in this case FMDV) that will infect a sus-ceptible cell in 50% of laboratory experiments.

Air sampling intercomparison

Air samples were taken with a Cyclone, Porton, May and the SKCBioSampler; a summary of the attributes of these is given at Table 1 .Sampling times and ow rates, selected on past experience, were varied inorder to determine the optimum sampling conguration. Air samples weretaken daily from 14 d.p.i. in the loosebox and also on days 2, 3 and 4 p.i.from two pigs, randomly selected on each sampling day and placed in a610-L cabinet (Gibson and Donaldson, 1986 ); the cabinet was thoroughlydisinfected with FAM (Evans Vanodine International) between eachanimal sampling. Daily variations in virus production were anticipated sothat instruments were compared sequentially, within minutes of eachother.

In the loosebox, the Porton and BioSampler were operated at 11 L/minfor 5 and 10 min. In addition a 20 min sample using the BioSampler wasalso taken; a 20 min Porton sample was not possible due to the loss of sampling medium created by the high level of evaporation from the

sampling chamber. In the cabinet, sampling time was restricted to 5 min to

allow for several samples to be taken and to ensure minimum discomfortto the pigs.

The May sampler was operated at 55 L/min for 5, 10 or 20 min in theloosebox and for 5 min in the cabinet. The Cyclone sampler was operatedfor 5, 10 or 20 min at ow rates of 390, 570 and 750 L/min; due to the highairow it was only possible to sample air from the loosebox using theCyclone.

The sample collecting uid was MEM-HEPES with added antibioticsand BSA (Alexandersen and Donaldson, 2002 ), at pH 7.2. All sampleswere stored at 70 C before being assayed for both virus infectivity andviral RNA genome.

Virus isolation

The infectivity of the impinger uid collected from the air samples wasassayed by inoculation of primary cultures of BTY cells ( Snowdon, 1966).Tenfold dilution series of collecting uid samples were made and eachdilution was inoculated onto three tubes. Titres were calculated by theKarber formula according to Lennette (1964) . The concentration of virusper litre of air was determined by the end-point titration of virus in eachcollected sample multiplied by the volume of the collecting uid anddivided by the ow rate of the sampler.

ELISA

An anigen ELISA ( Hamblin et al., 1984 ) was used to conrm thespecicity of the cytopathogenic effect observed in cell cultures inoculatedwith air samples. Serum samples were tested by an ELISA for antibodiesto FMDV ( Hamblin et al., 1986 ).

RNA extraction

QIAamp MinElute Virus Spin kit (Qiagen) was used for RNAextraction from air samples according to the manufactures instructions.Briey, RNA was extracted from 200 l L of the original samples and anal volume of 18 l L was recovered at the end of the process. This

material was used for RT-PCR. An automated extraction procedure wasused for nucleic acid extraction of serum samples ( Reid et al., 2003).

Quantitative real-time reverse transcription PCR (RT-PCR)

These were performed as previously described, including the primersand probe used in Experiment C of Quan et al. (2004) . The PCR wasperformed on a Strategene MxPro 3005P QPCR System. Results wereanalysed by means of MxPro QPCR software and genome copy numbersper reaction were also calculated according to Quan et al. (2004) . Theconcentration of genome copy numbers per litre of air was determined bythe quantity of virus genome per millilitre in each collected sample mul-tiplied by the volume of the collecting uid and divided by the ow rate of the sampler.

Results

Evidence of infection

Typical FMD signs were registered in all pigs approxi-mately 12 days after challenge. Generalised lesions wereobserved in all animals and, for humane reasons, pignumber VL 38 was euthanised after sampling on 2 d.p.i.,followed by VL 33, VL 34 and VL 35 on 3 d.p.i. Theremaining pigs, VL 36 and VL 37, were kept until theend of the experiment at 4 d.p.i.

Serum samples analysed by quantitative real-time RT-

PCR indicated that viraemia initiated at 1 d.p.i. ( Fig. 1),

220 C.M.F. Amaral Doel et al. / The Veterinary Journal 179 (2009) 219224


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reached peak levels at 2 d.p.i. and started to decline ataround 3 d.p.i. No antibodies were detected in any of thepigs throughout the sampling period. It is known that anti-bodies against FMD are detectable in serum shortly afterthe cessation of viraemia and correlate well with the endof viral excretion ( Sellers et al., 1977; Gibson and Donald-

son, 1986).

In summary the results suggested that infection followedan expected pattern and that these conditions provided anexcellent basis for the instrument intercomparison. Basedon this evidence and past experience, FMDV aerosol wasexpected to take place from 14 d.p.i. Due to the severityof disease and the need to adhere to strict animal welfarecriteria the usefulness of some of the samples from theair cabinet was a little reduced; some animals had to beeuthanised, for humane reasons, before the end of theexperiment.

Air sampling intercomparison

May and BioSamplerTable 2 shows the results obtained when, sampling from

the loose box, the May and the BioSampler were com-pared. The May sampler was the only one to detect infec-tious particles on 1 d.p.i., whereas both samplers detectedinfectious particles on 2 d.p.i. All samples collected on 3and 4 d.p.i. were negative for infectivity. In relation toTCID 50 per litre of air, the May detected the highest infec-tious doses during a 5 min sampling regime on 1 d.p.i. and,on 2 d.p.i., sampling for 10 min gave a marginally highernumber of TCID 50 per litre of air than sampling for 5 or20 min.

When analysing the copy numbers of FMDV RNA

extracted from the samples, our results indicated that the

Table 1Characteristics of the air sampling instruments used in this study

Porton BioSampler May Cyclone

Principle of operation

Liquid impinger Liquid impinger Three stage impactor Particles impacted on side of aglass chamber and washed byimpinger uid

Particle sizing


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best sampler was the BioSampler on 1 d.p.i. when samplingfor 5 min. As observed with infectivity, the 5 min sampling

time generated a higher number of copies per litre of air onthe majority of the collection days than all other samplingtimes examined here. On 3 d.p.i., the sample collected dur-ing 20 min gave the single positive result for that day. How-ever, on 4 d.p.i., it was the 10 min sampling time whichproduced the only two positive samples for this day whencollected by both the May and the BioSampler. Peak levelswere recorded at 1 d.p.i. with samples collected with theBioSampler.

May and PortonWhen the performances of the May and the Porton sam-

plers were compared for sampling in the loose box, results

suggested that the highest infectivity was recovered on2 d.p.i. (Table 3 ). On 1 d.p.i. both samples collected bythe May sampler gave positive results but the samples col-lected by the Porton sampler were both negative for infec-tivity. All samples from 2 d.p.i. were positive for infectivitywhereas all samples collected during 3 and 4 d.p.i. withboth the May and the Porton were negative for infectivity.The optimum sampling time seemed to be 5 min for bothsamplers.

Detection of FMD viral RNA correlated with detectionof infectivity for samples collected during 1 and 2 d.p.i. On3 d.p.i., sampling for 5 min with both samplers generated

the only two positive samples for viral RNA. However,on 4 d.p.i. only a single positive sample was observed; this

was collected with the Porton sampler for a period of 10 min.Results obtained with samples collected from the sam-

pling cabinet on 2, 3 and 4 d.p.i. were broadly similar tothose obtained with the loose box and indicated that theMay gave the highest TCID 50 when its performance wascompared with the BioSampler and the Porton (resultsnot shown).

CycloneTable 4 summarises results obtained when the perfor-

mance of the Cyclone sampler was evaluated by applyingdifferent ow rates per minute while collecting samples.

The results show that, during 1 d.p.i., samples collectedfor 5 min at a ow rate of 570 (total of 2850 L) recoveredthe highest TCID 50 as well as the highest copy numbersof genome per litre of sample than the samples collectedfor 10 or 20 min at this ow rate. No other ow rates weretested during 1 d.p.i. On 2 d.p.i., signicantly higher num-bers of infectious particles and virus genome were observedfrom samples where a ow rate of 390 L/min was appliedfor 5 min. For 3 d.p.i., recovery of infectious particleswas marginally greater at 3750 or 5700 L, where identicalresults were observed. However, in relation to virus gen-ome, it was once again the 1950 ow rate that generated

Table 2Comparison between the May and the BioSampler

Duration of sampling (min)

Equipment Total numberof litres of air

Flow rate(L/min)

D1 p.i. D2 p.i. D3 p.i. D4 p.i.

TCID 50 /La

Copies/Lb

TCID 50 /La

Copies/Lb

TCID 50 /La

Copies/Lb

TCID 50 /La

Copies/Lb

5 May 275 55 1.09 2841.66 0.11 3129.30 0 0 0 0

BioSampler 55 11 0 7249.35 0.99 3407.65 0 0 0 010 May 550 55 0.12 145.89 0.12 1624.84 0 0 0 92.13

BioSampler 110 11 0 1001.70 0 2938.03 0 0 0 919.04

20 May 1100 55 0.35 681.72 0.03 353.25 0 251.95 0 0BioSampler 240 11 0 1031.83 0 671.25 0 0 0 0

Samples were taken in the loosebox during 5, 10 and 20 min.D: day, p.i.: post-infection.

a TCID 50 /L: as isolated in BTY cells.b Copies/L: number of genome copies per litre of sample.

Table 3Comparison between the May and the Porton samplers

Duration of sampling (min)

Equipment Total numberof litres of air

Flow rate(L/min)

D1 p.i. D2 p.i. D3 p.i. D4 p.i.

TCID 50 /La

Copies/Lb

TCID 50 /La

Copies/Lb

TCID 50 /La

Copies/Lb

TCID 50 /La

Copies/La

5 May 275 55 0.35 779.36 15.51 27426.32 0 1873.02 0 0Porton 55 11 0 0 9.51 8069.87 0 2,06,518 0 0

10 May 550 55 0.05 293.81 4 11,690 0 0 0 0Porton 110 11 0 0 7.49 2815 0 0 0 454.25

Samples were taken in the loosebox during 5 and 10 min.D: day, p.i.: post-infection.

a TCID 50 /L: as isolated in BTY cells.b

Copies/L: number of genome copies per litre of sample.



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the greatest number of copies per litre. All samples col-lected on 4 d.p.i. were negative for both infectivity and viralRNA.

Discussion

In the event of an outbreak of disease, an accurateassessment of the risk of secondary infection, including air-borne transmission, is required by those responsible forcontrolling and eradicating the disease. This assessment isaided by a detailed knowledge of how much virus is likelyto have been produced by the infected livestock and this isunderpinned by the quality of measurements made underlaboratory conditions. Consequently, it is important toknow which instruments are the best at detecting the virus,and their limitations. This is supported by the requirement

to have optimised and documented standardised labora-tory analysis techniques. In addition as new instrumenta-tion becomes available a baseline against which they canbe compared is believed to be essential.

Based on the results from this study it is concluded thatin general there is no optimum air sampling techniquewhich could be successfully employed for all situationsbut that Cyclone type samplers are, perhaps the most effi-cient. This view is supported by Cox (1987) and Mouille-seaux (1990) who used the same type of instrument tomeasure other aerosols. All samples collected by theCyclone sampler on 1, 2 and, in particular 3 d.p.i. werepositive for infectivity suggesting that this is far the mostefficient sampler in recovering infectious particles thanany other sampler studied here. With regard to sampletime, there appeared to be little merit in taking samplesfor longer than 5 min. However, whilst in an enclosed roomthis may be optimal, it must be remembered that underother circumstances, e.g. in a larger unit or even out of doors it may be advantageous to sample for much longer,thus increasing the possibility of detecting low concentra-tions of virus. On the other hand, there is a possibility thatextended sampling may cause a decrease, or even loss, of infectivity and/or copy numbers of virus genome.

The May sampler was the best in relation to recovery of

infectious FMDV particles when compared with both the

Porton and the BioSampler. The May sampler has an addi-tional characteristic which has not been evaluated duringthis experiment; it allows the aerosol sample to be dividedinto three size groups (>6 l m, 36 l m and


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this will involve a number of signicant additional chal-lenges to those experienced above. For example, relativehumidity and temperature, which can inuence aerosol sur-vival are rigorously controlled in our animal isolation unitsbut are not so easily controlled in the eld ( Donaldson,1972). The development of a disposable sampler would also

be an advantage to make eld measurements a possibilityin the future.

Acknowledgements

The authors would like to thank the staff responsible forthe animal isolation unit 8 at IAH for their careful manage-ment. We are grateful to David Paton for his constructivecomments. This work was funded by the Department forEnvironment, Food and Rural Affairs (DEFRA), projectSE2926.

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