Foot-and-mouth disease: A review of intranasal infection of cattle, sheep and pigs

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The Veterinary Journal 177 (2008) 159–168

TheVeterinary Journal

Review

Foot-and-mouth disease: A review of intranasal infectionof cattle, sheep and pigs

Robert Sellers a, John Gloster b,*

a 4 Pewley Way, Guildford, Surrey GU1 3PY, UKb Met Office, based at the Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, Guildford GU24 0NF, UK

Accepted 15 March 2007

Abstract

In an outbreak of foot-and-mouth disease (FMD) it is important to identify animals at risk from airborne virus. Investigations havebeen carried out over the years to determine the dose required to infect cattle, sheep and pigs by the intranasal route. This paper reviewsthe results of investigations for animals which have been infected by instillation or spraying a virus suspension into the nostrils or byexposure to affected animals through a mask or by indirect contact.

The lowest doses were found by use of a mask. With virus from affected pigs given through a mask, doses of 18 infectious units (IU) incattle and 8 IU in sheep were found to cause infection and give rise to lesions. Overall, cattle required the least amount of virus followedby sheep. Pigs required a dose of 22 IU to cause infection and a dose of 125 IU to give rise to lesions. In many experiments pigs failed tobecome infected. With all three species the dose varied with the individual animal and the virus strain. For modelling previous outbreaksand in real time, a dose of 8 IU or 10 and 50% infectious doses (ID50) could be used where cattle and sheep were involved. Experience inthe field, combined with the results from experiments involving natural infection, indicate that pigs are not readily infected by the intra-nasal route. However, for modelling purposes a dose of about 25 IU should be used with care.

Investigations are needed to determine doses for virus strains currently in circulation around the world. In addition, the nature of theaerosol droplets needs to be analysed to determine how the respective amounts of infective and non-infective virus particles, host com-ponents and, in later emissions, the presence of antibody affect the survival in air and ability to infect the respiratory tract. Further workis also required to correlate laboratory and field findings through incorporation of the doses into modelling the virus concentrationdownwind in order that those responsible for controlling FMD are provided with the best available assessment of airborne spread.Finally, the doses found for infection by the intranasal route could be applied to other methods of spread where virus is inhaled to assessrisk.Crown Copyright � 2007 Published by Elsevier Ltd. All rights reserved.

Keywords: Foot-and-mouth disease; Intranasal infection; Review of experimental results

Introduction

Foot-and-mouth disease (FMD) is a highly infectiousviral disease of cloven-hoofed animals both domestic andwild. The disease spreads by contact between infectedand domestic animals, by animal products (milk, meat andsemen), by mechanical transfer on people, wild animals

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and birds, by vehicles and fomites and by the airborne route.Spread by airborne carriage on the wind has been considereda possibility from the beginning of the 20th century especiallyby Scandinavians (Bang, 1912; Donaldson, 1979; Penberthy,1901) and its part in spread of disease was recognised in theUK in the 1967–1968 and 2001 epidemics and in the spreadof FMD from Brittany to the Isle of Wight in 1981 and inHampshire in 1967 (Donaldson et al., 1982; Gloster et al.,2003, 2005a,b; Hugh-Jones and Wright, 1970; Mikkelsenet al., 2003; Sellers and Forman, 1973).

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160 R. Sellers, J. Gloster / The Veterinary Journal 177 (2008) 159–168

The airborne disease cycle can be divided into threestages: emission, transport and inhalation. Before 1984,the amounts of virus emitted by infected animals, the sizeof the infective particles and their survival in air was gener-ally established but the process of inhalation, especially thedose required to initiate infection by the respiratory route,remained to be investigated more fully. A review of aspectsof airborne spread of FMD is given in the paper on path-ogenesis and diagnosis of FMD by Alexandersen et al.(2003b).

The potential for the spread of FMD by the airborneroute can be determined by estimating the amount of virusreleased into the atmosphere and establishing the meteoro-logical conditions in the vicinity of infected animals. Thisinformation is used as input into an atmospheric dispersionmodel which calculates downwind concentrations of virusand an inhaled dosage (Gloster et al., 2003; Ryall and Mar-yon, 1998; Sørensen et al., 2000, 2001). To determine thearea at risk from airborne infection the dosage to initiateinfection is also required.

This paper provides a review of the published experi-mental data involving cattle, sheep and pigs with a viewto giving guidance on dosage to those required to modelthe airborne risk of disease spread and to those responsiblefor controlling disease outbreaks. The dose determined canalso be used to assess the risk of infection of the respiratorytract, where virus is inhaled from contact with affected ani-mals, contaminated personnel, vehicles that have previ-ously transported affected animals, aerosols from milkspills and from contaminated fomites.

Experimental conditions

The experiments can be divided into four categories:-

1. Application by instillation of virus suspension into the nos-

trils – artificial method of infection with artificially pre-pared virus.

2. Application by a spray of virus suspension to nostrils –artificial method of infection with artificially preparedvirus.

3. (a) Exposure of animals to the aerosols of infected

animals through a mask – artificial method of infectionwith naturally produced virus. (b) Exposure of cattle

virus generated from a May spinning top through a mask –artificial method of infection with artificially preparedvirus.

4. Indirect contact between infected animals and clean reci-

pient animals – natural method of infection with natu-rally produced virus.

All of the experiments were carried out in isolation unitsunder disease controlled conditions with animals fromEuropean breeds up to 3 years old. FMD strains of allvirus types except SAT1 and SAT3 were used. The typeof spray and the collecting apparatus varied betweenexperiments.

FMD virus was titrated by different methods: intra-dermal inoculation of the tongues of cattle (Gravesand Cunliffe, 1960; Henderson, 1952; Korn, 1957),unweaned mice (Eskildsen, 1969; Terpstra, 1972), sheep,lamb and pig kidney monolayer tissue cultures (Sutmol-ler et al., 1968; McVicar and Sutmoller, 1976, 1969;Bouma et al., 2004; Brown et al., 1992, 1996) andbovine thyroid monolayer (BTY) tissue cultures, whichfrom 1981 onwards were used in the majority of exper-iments. BTY cultures had been found to be the mostsensitive at detecting virus in air samples from affectedanimals (Donaldson et al., 1970). Strains adapted topigs do not grow in BTY cultures and the pig kidneyIBRS2 cell line was used instead (Dunn and Donaldson,1997). In experiments from 2002 onwards, viral RNA invirus containing material was measured by the reverse-transcriptase polymerase chain reaction (RT-PCR)method.

The genome equivalents per millilitre were about100–1000-fold higher than the titre of infective virus inBTY cells, although in late infection this increased upto 100-fold more, probably due to the presence of anti-bodies (Alexandersen et al., 2003a). This ratio betweengenome equivalents and infective virus is similar to theratio of non-infective 25 nm particles and infective25 nm particles (100–1000-fold to 1) found by electronmicroscopy (EM) of virus suspensions (Report, 1956-1960; Bradish et al., 1960).

Evidence of infection was taken as the presence of clin-ical signs, viraemia and seroconversion, or viraemia andseroconversion, or viraemia or seroconversion alone.Some FMD strains do not give rise to detectable lesionsin cattle, pigs and especially in sheep (Alexandersenet al., 2003b). Viraemia can be of short duration withoutleading to the development of antibody (Garland, 1974;Gibbs et al., 1975; Donaldson and Kitching, 1989). Whereanimals were killed during the incubation period, thepresence of virus in more tissues than earlier in the incu-bation period was taken as evidence of infection. Animalsmay also show transient levels of antibody (Alexandersenet al., 2002; Alexandersen and Donaldson, 2002). Animalswith transient antibody levels 1 in 45 or greater wereregarded as being infected. Animals without detectablelesions may pass on infection to others (Callens et al.,1998).

Review of animal experiments

The experimental results by all methods of infection forcattle, sheep and pigs are given in Tables 1–3 and summa-rised in Table 4 and Fig. 1. Experiments specificallydesigned to estimate dose are indicated in the tables (D).The remaining experiments were part of investigations onthe pathogenesis of FMD, the exposure of vaccinated ani-mals to infection and the development of carriers, in whichthe dose was also measured.

Table 1

Recipient animals – cattle

Method Virus type/strain

Virussource

Dose (logIU) Amount Experimentallength

C V S +ve �ve No.Fig. 1

Comments

Instillation A4691 2.0 PK/SK IU 5 mL suspension 2 2 2 4 Sutmoller et al. (1968)4.0 PK/SK IU 5 mL suspension 2 2 2 2 06.0 PK/SK IU 5 mL suspension 2 2 2 2 0

Instillation 01 3.0 PK/SK IU 5 mL suspension 1 1 1 1 1 McVicar and Sutmoller (1976)4.0 PK/SK IU 5 mL suspension 2 2 2 2 05.0 PK/SK IU 5 mL suspension 4 4 4 4 07.0 PK/SK IU 5 mL suspension 3 3 3 3 0

Instillation O/NET/2001

3.0 Cattle IU(=3.8 PK IU, 3.6LK IU)

3 mL suspension 3 5 6 6 0 Bouma et al. (2004). No end point

Spray 039 D 5.1 Cattle IU 2 mL spray 6 6 2 Henderson (1952)

Spray A119 D 1.95 Cattle IU 2 mL spray 2 2 4 Henderson (1952)4.95 Cattle IU 2 mL spray 2 2 06.95 Cattle IU 2 mL spray 2 2 0

Spray AM1 D 5.85 Cattle IU 3 · 4 mL spray 1 1 1 Henderson (1952)

Spray O1BFS1860

0.8 BTY IU 0.01 mL fine spray 0/1?

3 Burrows et al. (1981) 1? ?contactinfection

3.1 BTY IU 0.3 mL coarsespray

4 2 4 0

3.5 BTY IU 0.01 mL fine spray 3 3 0 Virus in tissues in incubation period5.75 BTY IU 0.3 mL coarse

spray3 3 0

Spray O 4.0 Mouse IU 100 mL spray 1 0 Eskildsen (1969)5.0 Mouse IU 100 mL spray 1 0 Virus in tissues in incubation period5.0 Mouse IU 100 mL spray 1 1 0 No end points5.7 Mouse IU 100 mL spray 1 1 06.2 Mouse IU 100 mL spray 1 1 0

Spray O2 3.6 Cattle IU 0.5 mL spray 4 0 Korn (1957). Virus in tissues at 63 hp.i. No end point

Spray A 8.3 LK IU 1 mL spray 1 1 0 Brown et al. (1996). No end point

Spray Asia1 5.8 LK IU 2 mL spray 3 3 0 Brown et al. (1992). No end point

Mask O1BFS1860D

Aerosol 3.85 to 5.05 BTYIU

1–1.5 min 5 5 6 6 0 1 Donaldson et al. (1987)

from 1.25 to 2.45 BTYIU

1–2 min 4 4 6 6 0 No end point

May 0.95, 1.25, 1.45,2.25

1 min 2 2 4 4 No end point

spinning BTY IUtop 1.05, 1.25 BTY

IU1 min 2

Mask SAT2 SAR3/79D

2 pigs 1.45–1.75 BTYIU

10 min 5 5 5 5 0 1a Donaldson et al. (1987)

1.25, 1.55, 1.95BTY IU

10 min 2 3 2 3

1.45, 1.55 BTYIU

10 min 2

1.45, 1.6, 2.25BTY IU

5 min 3 3

1.55, 1.55 BTYIU

5 min 2

Indirect O1BFS1860

2 pigs 2.25 BTY IU 60 min 3 3 3 0 2 Donaldson and Kitching (1989)2.35 BTY IU 60 min 3 3 3 0 No end points3.25 BTY IU 60 min 3 3 3 04.25 BTY IU 60 min 3 3 3 0

Indirect OUKG2001

3 pigs 3.85 BTY IU 2 h 1 1 1 1 1 3 Aggarwal et al. (2002)

Indirect OUKG2001

4 sheep N/A 5 h 2 1 1 2 0 Aggarwal et al. (2002). No dosegiven

Key: C = clinical signs, V = viraemia, S = seroconversion or carrier; PK = pig kidney monolayer tissue cultures, SK = sheep kidney monolayer tissue cultures, LK = lamb

kidney monolayer tissue cultures, BTY = bovine thyroid monolayer tissue cultures, IU = infectious units, p.i. = post infection, trans = transient, N/A = not available. The

dose is the amount of virus inhaled at the nostrils, D = experiment to determine dose, ID50s are expressed as IU (ID50 · 0.7 assuming a Poisson distribution). The number in

the penultimate column (no. Fig. 1) cross refers to the X-axis in Fig. 1.

R. Sellers, J. Gloster / The Veterinary Journal 177 (2008) 159–168 161

Table 2Recipient animals – sheep

Method Virustype/strain

Virussource



Comments

Instillation O Greece23/94

4.9 BTY IU 2 mLsuspension

14 15 15 16 0 Hughes et al. (2002). No endpoint

Instillation O2 4.0 PK/SK IU 0.2 mLspray

3 1 McVicar and Sutmoller(1969)

Instillation A1 4.0 PK/SK IU 0.2 mLspray

4 0 McVicar and Sutmoller(1969). No end point

Instillation A4691 4.0 PK/SK IU 0.2 mLspray


Instillation CTdF 4.0 PK/SK IU 0.2 mLspray


Instillation O Greece23/94


0 5 Hughes (2002)


1 1 4


4 5 5 0


4 5 5 0


4 5 5 0

Mask O1 BFS1860 D

2 pigs 1.8, 2.1, 3.0BTY IU 0.45

15 min 3 3 3 3 4 Gibson and Donaldson(1986)

1.3 BTY IU 15 min 20.9, 0.9, 1.3, 1.8BTY IU

10 min 2 4 4 4

0.95 BTY IU1.8, 1.9, 2.0BTY IU

10 min 1

0.7,1.7 BTY IU 10 min 1 3 3 31.7, 2.1, 2.3BTY IU

10 min 2

1.0 BTY IU 10 min 2 3 3 30.9, 0.9, 0.9, 1.0,1.7 BTY

10 min 1

IU 10 min 5 5 5 5 0

Indirect O1 BFS1860

4 pigs 3.05 BTY IU 2 h 4 4 4 4 0 7 Gibson et al. (1984). No endpoint

Indirect O UKG2001

3 pigs 3.45 BTY IU 2 h 3 4 4 4 0 8 Aggarwal et al. (2002). Noend point

Indirect O UKG2001

4 sheep N/A >5 h 4 4 5 5 0 Aggarwal et al. (2002)No end pointNo dose given

Indirect O UKG2001 D

3 sheep 1per box

2.35 BTY IU 24 h 2 2 1 5 Esteves et al. (2004)

Indirect O UKG2001

2 sheep 3.15 BTY IU 2 h 0 2 6 J.-F. Valarcher et al.,unpublished data

3.45 BTY IU 4 h 0 2 No end points3.65 BTY IU 6 h 0 23.75 BTY IU 8 h 0 24.25 BTY IU 25 h 0 2

For key see Table 1.


Cattle

Most experiments were carried out with type O strains,but type A, SAT 2 and Asia 1 strains were also used. Insome successful experiments no end point was determined

(Table 1). The lowest dose by instillation was found to belog2.0 IU (100 IU) (Sutmoller et al., 1968) and by spraywas log1.95 IU (90 IU) (Henderson, 1952). In experimentsusing a mask, log1.25 IU (18 IU) from natural infectionand log0.95 IU (9 IU) from spray from a May spinning

Table 3Recipient animals – pigs

Method Virustype/strain

Virussource



Comments

Instil O D >6.25 CattleIU

1 mL 0 2 Graves and Cunliffe (1960). Noinfection or end point

Spray O1 3.95 Mouse IU 2 mL 2 0 Terpstra (1972). Virus in tissues at 72and 96 h p.i., no end point

Mask O1 Laus 2 pigs 1.35, 1.95, 2.1,2.45, 2.45,

10 min 1 7 7 9 Alexandersen et al. (2002) 2/7antibodies at 14, but not 21 dpi.

SW/65 2.45, 2.55 BTYIU 1.95,

D 2.35 BTY IU 10 min 2

Mask O1 Laus 3 pigs 1.25, 1.35,1.35, 1.55,

2 and 10 min 0 7 9 Alexandersen et al. (2002)

SW/65 1.55, 2.05, 2.1BTY IU

No end point

D

Mask O1 Laus 3 pigs 1.35, 1.55, 1.75BTY IU

10 min 0 3 9 Alexandersen et al. (2002)

SW/65 No end pointD

Mask O1 Laus 3 pigs 1.35, 1.45,1.95, 1.95,


SW/65 2.85 BTY IU No end pointD

Mask O1 Laus 3 pigs 1.35, 1.95,2.45, 2.45, 2.6


SW/65 BTY IU No end pointD

Mask O1 Laus 3 pigs 1.55, 2.85, 3.1,3.1, 3.45

5 min 1 5 5 10 Alexandersen and Donaldson (2002) 4trans antibodies

SW/65 BTY IUD 1.45, 2.95, 3.35

BTY IU5 min 3

Mask O SKR 3 pigs 2.25, 2.25,2.35, 2.35,

5 min 0 10 10a Alexandersen and Donaldson (2002)

1/2000 2.55, 2.55,2.85, 2.95,

No end point

D 3.25, 3.25 BTYIU

Indirect O UKG2001

3 pigs >4.7 BTY IU 24–48 h 0 8 10b Alexandersen and Donaldson (2002) 1trans antibodyNo end point

Indirect O UKG2001

3 pigs 3.1 BTY IU 2 h 0 4 11 Aggarwal et al. (2002)No end point

Indirect O UKG2001

4 sheep N/A 5 h 4 4 4 0 Aggarwal et al. (2002)No end point

Indirect O Taw9/97

1 pig in 4boxes

>3.25 BTY IU 24–48 h 0 8 12a Alexandersen et al. (2003a) 1 trans

antibodyNo end point

Indirect C NovSW 73

1 pig in 4boxes

>5.1 BTY IU 24–48 h 0 8 12 Alexandersen et al. (2003a)No end point

For key see Table 1.


top (Mitchell and Stone, 1982) were the lowest doses. Thelowest dose to cause clinical lesions was log1.25 IU (18 IU)after natural infection or spraying through a mask. Thehighest dose that failed to cause infection using a mask

was log1.55 IU (35 IU) for natural infection andlog1.25 IU (18 IU) from spray from a May spinning top(Donaldson et al., 1987). The lowest dose for indirect con-tact infection was log2.25 IU (180 IU) (Donaldson and

Table 4Minimum dose (IU) to initiate sub-clinical and clinical infection: highest dose to fail to cause infection

Instillation Spray Indirect contact Mask

1 2 3 1 2 3 1 2 3 1 2 3

Cattle 100 90 180 180 7100 9 18 35Sheep 2250 7100 2250 1120 1120 17,800 8 8 50Pigs >1,800,000 9000 >250,000 >250,000 250,000 22 125 2250

1, minimum dose to initiate sub-clinical infection; 2, minimum dose to initiate clinical infection; 3, highest dose not to initiate infection.


Kitching, 1989). Henderson (1952) found a differencebetween strains for the lowest dose to cause infection(Table 1), a finding that he correlated with differences inreaction to contact infection. In the experiments withmasks the dose to cause infection varied with individualanimals.

Sheep

Strains of types O, A and C were used in the experimentsthe majority being of type O. End points could not be dem-onstrated in every experiment, some showing successfulinfection, one other failure to infect by indirect contact(J.-F. Valarcher et al., unpublished data). The lowest dosefor instillation was log3.35 IU (2250 IU) (Hughes, 2002).In mask experiments the lowest dose was found to belog0.9 IU (8.0 IU), which was also the dose to cause clini-cal infection. The highest dose that failed to cause infectionwas log1.7 IU (50 IU) (Gibson and Donaldson, 1986). Inexperiments where sheep were exposed by indirect contact

6

5

4

3

2

1

0

Log

IU

No infection Infection No infection

Key: Numbers = reference identification (Tables 1 to 3

* = Spinning top

1 1a 3 1 1a 2 3 4 5 6

M M I M M I I M I I * P P * P P P P S S

Cattle She

Fig. 1. Summary of results from experiments which involved exposure of cattlethrough a mask or by indirect infection. The results from experiments involvinthe source and method of administering the virus is given (C = cattle, P = pig, San experiment animals have been recorded as non-infected and infected the saexample Gibson and Donaldson (1986) exposed sheep to virus from a pig (P), uwhich became infected (filled in squares) and others which remained uninfecte

a dose of log2.35 IU (225 IU) over 24 h was found to infect(Esteves et al., 2004). Differing values were found in thedose required to infect individual sheep after natural infec-tion through a mask.

Pigs

The strains used in the experiment were of type O apartfrom C Noville. Instillation of log6.25 IU (1,800,000 IU)failed to give rise to lesions in pigs (Graves and Cunliffe,1960). Virus was recovered from the tissues of pigs killed72 and 96 h after receiving a dose of log 3.95 IU(9000 IU) (Terpstra, 1972). The lowest dose to cause infec-tion in pigs exposed to natural virus through a mask waslog1.35 IU (22 IU), while the lowest dose to cause lesionswas log2.1 IU (125 IU) (Alexandersen et al., 2002). Thehighest dose to fail to cause infection after exposure to nat-ural infection through a mask was log3.35 IU (2250 IU)(Alexandersen and Donaldson, 2002). Experiments wherepigs were exposed to natural infection with naturally pro-

Pigs

Infection No infection Infection

), M = Mask, I = Indirect, P = Pig, S = Sheep,

4 7 8 5 9 10 10a&b 111212a 9 10

M I I I M M M I I I I M M P P P S P P P P P P P P P

ep

, sheep and pigs to artificially prepared virus through a mask, natural virusg cattle, sheep and pigs have been plotted separately. For each experiment

= sheep. * = spinning top, M = mask and I = indirect contact). Where inme symbol has been plotted (open = no infection, closed = infection). Forsing a mask (M) and estimated the dosage for a number of animals some ofd (open squares).


duced virus by a natural method of infection – indirect con-tact – were not successful (Alexandersen and Donaldson,2002; Aggarwal et al., 2002; Alexandersen et al., 2003a).

There was a difference between strain O UKG2001 andstrain O SKR 1/2000 in the successful response and failureto respond respectively to infection through a mask by nat-ural virus (Alexandersen and Donaldson, 2002). In themask experiments there was variation in the responses ofindividual pigs to the doses given (Alexandersen et al.,2002; Alexandersen and Donaldson, 2002).

It can be seen in Fig. 1 that the lowest dose was foundusing a mask (artificial method, natural virus). There waslittle difference between cattle, sheep and pigs in the lowestdose to cause infection; however with pigs a greater dosethan that for cattle was found to give rise to lesions. Inaddition a dose 60-fold greater than that for cattle failedto infect pigs (Alexandersen and Donaldson, 2002). Withinstillation, spray and indirect contact the doses found toinfect cattle were considerably less than the doses requiredto infect pigs. Sheep also required a higher dose than cattlewhen infection was by instillation or indirect contact(Table 4).

Discussion

It may be thought that the absence of any standardizedtechnique and the differences in housing, instrumentationand methods of measurement make it impossible to drawconclusions about the dose required by the intranasalroute. However, in the majority of experiments reportedfrom 1981 onwards, BTY tissue cultures (the most sensitivesystem) were used for virus assay. In addition, such exper-iments were reported from one centre (BBSRC, Pirbright),where from 1960 infected livestock were held in units undernegative pressure, although the nature of the ventilationsystem changed over the years.

Instillation and spraying of virus preparations into thenostrils were the methods used to measure the dose in ear-lier experiments in cattle. In cattle the lowest doses byinstillation (100 IU, Sutmoller et al., 1968), spray (90 IU,Henderson, 1952) and also by coughing and sneezing(100 ID50, Sellers et al., 1971) were similar. They were alsolower than the doses found for pigs (Graves and Cunliffe,1960 – instillation; Terpstra, 1972 – spray). In later exper-iments the development of the use of a mask ensured thatvirus from natural sources was introduced to the respira-tory tract more effectively and the doses for cattle and pigswere found to be lower than those found by instillation andspray. As with instillation and spray in the earlier experi-ments the dose for cattle by administration through a maskwas lower than that for pigs. In the field pigs are less likelyto be infected through airborne spread than cattle. Thedoses used in previous modelling were based on thosefound in early experiments (Gloster et al., 1981; Donaldsonet al., 1982). In later modelling, the lowest doses found incattle and sheep by mask experiments (natural infectionby artificial means) were used.

In investigations of doses of FMD virus strains in thefuture the use of a mask (artificial method of infection withnaturally produced virus) would be the method of choice.The method most resembling that in the field (naturalmethod with natural virus – indirect contact) did not resultin lower doses and higher doses were found by artificialinfection (instillation and spray). It should be pointed outthat the majority of experiments by instillation, spray andindirect contact were not carried out to measure dose.The experimenters were using the method they found mostappropriate to infect the animals.

The dose to initiate infection varied with the virus strainused and between individual animals. Some of this varia-tion could be attributed to the fact that experiments werecarried out under different conditions and with differingassay systems. Overall slight differences could be importantin the field, where at low doses some highly susceptibleindividuals could become infected, whereas with otherstrains other individuals may escape infection resulting inthe failure of disease spread.

Where exposure was by indirect contact (natural methodand natural virus) factors in addition to animal species,individual animal and virus strain affected the success ofinfection by different doses. Such factors included the con-struction of the building and the airflow in the isolationunit. Successful infection by indirect contact was achievedin some of the experiments described here as well as byFogedby et al. (1960), Burrows (1968), Garland (1974)and Sellers et al. (1968). Failure to cause infection in someof the experiments reviewed here was found by other inves-tigators, such as Traub and Wittman (1957), where infec-tion was directed from infected animals in a shed tocalves and pigs. Bouma et al. (2004) found no evidenceof infection when calves were exposed by indirect or directcontact to calves infected and reacting to the FMD strainof the 2001 Netherlands outbreak. However the lesser sus-ceptibility of calves to the Netherlands virus as well as theenvironment could have been responsible for the failures.

Hutber and Kitching (2000) analysed the spread ofFMD within a cattle herd in Saudi Arabia and concludedthat spread had occurred through aerosol between penshousing calves. This report also emphasized the importanceof spatial or physical barriers in preventing cross-infection.Differences in inhalation of FMD virus by people in exper-iments in different laboratories could be explained by thepresence or absence of air currents (Amass et al., 2003;Donaldson and Sellers, 2003). Air currents were found toassist the spread of viruses not known to cause airbornespread over distance, for example, African swine fever(ASF) and classical swine fever (CSF) (Dewulf et al.,2000; Wilkinson et al., 1977).

Successful infection in cattle and sheep occurred 1–10 min after doses of 8–18 IU given by mask. Sellerset al. (1970) found virus in the nose 5 min after contactwith affected pigs. This indicated that if virus is presentin the air it can be inhaled rapidly and give rise to success-ful infection in susceptible animals. Any virus particles that


fail to cause infection would be cleared from the respira-tory tract and unlikely to build up until a critical dose isreached.

Infective aerosol particles inhaled through the nostrilspass through the nares and thence to sites in the respira-tory tract, where the virus multiplies and spreads to otherparts of the body (Burrows, 1972). The site of initial mul-tiplication after inhalation of particles from naturalsources has been found to be the pharyngeal area (Bur-rows et al., 1981; Alexandersen et al., 2003b). In the exper-iments described in this paper, particles from artificialsources initiated infection in the nasal mucosa and lungas well as in the pharyngeal area. In some papers the dis-tribution of particles size between 2 and 10 lm is given,but owing to the range of sizes inhaled or given by sprayit is not possible to determine from the results which sizeof particle initiated infection and in which part of therespiratory tract.

Gloster et al. (2006) decided that there was no need totake into account the size of the particles when modellingspread of disease in the field. However the size of aerosolparticles and their site of initial infection in the respiratorytract are important in studying the pathogenesis of the dis-ease. In addition, knowledge of particle size and likely siteof infection are required for designing vaccines or antiviralsubstances for protection of the respiratory tract. The com-position of individual aerosol particles may be important indetermining the ability to initiate infection in the respira-tory tract. It could be assumed by correlation with EMresults that genome equivalents represent one infectivevirus particle to 100 or more non-infective particles. Anaerosol particle may contain virus particles infective forBTY tissue cultures, non-infective particles, host materialand, in the later stages of infection, antibody. Presence ofantibody and high content of non-infective particles emit-ted from animals later in the infection may impair the abil-ity to infect animals by the respiratory route. Furtherinvestigations of the nature of the aerosol particle arerequired.

Investigations have been made by modelling of pastFMD outbreaks in UK in the Isle of Wight and in the1967/68 and 2001 epidemics, where no cause other than air-borne carriage of virus could be identified. The virus con-centration at the site of the outbreak determined fromthe models was found to be up to 7000-fold less than theconcentration that would lead to infection in cattle basedon the dose of 10 TCID50 (Sørensen et al., 2000; Donald-son et al., 2001; Gloster et al., 2003, 2005a,b). The discrep-ancy between laboratory results and field findings could bedue to lack of information on the output and timing ofinfection in animals and transmission including meteoro-logical factors as well as to the dose inhaled. Finding thatcattle and sheep can be infected by doses less than 18 IUand 8 IU, respectively, is unlikely to increase the concentra-tion downwind significantly. Future research would be bet-ter directed at examining virus output and meteorologicalfactors. The meteorological process, procedures and fac-

tors involved have been discussed elsewhere (Glosteret al., in press). See reference section for latest situation.

The minimal doses derived from the experiments canalso be used to determine the risk through inhalation ofaerosols derived from secretions and excretions frominfected or contaminated animals, from contaminated peo-ple and contaminated vehicles, milk spills and fomites(Donaldson, 1979). The amounts of virus emitted in secre-tions and excretions are given in Sellers (1971), Thompson(1994) and Alexandersen et al. (2003b).

Conclusion

The experiments reported show that the lowest doses forcattle and sheep after natural infection were 18 IU and8 IU, respectively. These values could be used for model-ling airborne spread given the present state of knowledge.However investigations need to be carried out on the aero-sol particle itself to define not only its components but alsoits capability both for infecting the respiratory tract at dif-ferent stages of infection in the donor and for its survivalover distance. The discrepancy between virus concentra-tions based on laboratory measurements of output andconcentrations found downwind in field outbreaks indi-cates that further investigations on virus output and down-wind transport are required. The experiments, togetherwith field experience, indicate that spread within buildingsmay vary due to a number of factors. They raise questionsabout emission from and spread within buildings during anoutbreak. The doses determined can also be used in deter-mining the risk of aerosol spread in the field when methodsof spread such as via contaminated vehicles and fomites areinvolved.

Acknowledgements

The authors express thanks to former and present col-leagues at the Institute for Animal Health and the Met Of-fice. Defra are thanked for funding John Gloster’scontribution to preparation of this paper (contract SE2926). Alex Donaldson, Tony Garland and David Patonare thanked for providing very helpful comments on thedraft text.

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Documents

Foot-and-mouth disease: A review of intranasal infection of cattle, sheep and pigs