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Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with O1 and sat 2 strains
Research in Veterinary Science 1987, 43, 339-346
Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with 01 and SAT 2 strains A. 1. DONALDSON, Animal Virus Research Institute, Pirbright, Woking, Surrey, GU240NF, C. F. GIBSON, May and Baker Ltd, Rainham Road South, Dagenham, Essex, RMIO 7XS, R. OLIVER, Central Animal Health Laboratory, Ministry of Agriculture and Fisheries, Wallaceville Animal Research Centre, Private Bag, Upper Hutt, New Zealand, C. HAMBLIN, R. P. KITCHING, Animal Virus Research Institute, Pirbright, Woking, Surrey, GU240NF
Equipment has been constructed and methods developed for exposing individual cattle to two strains of foot-and-mouth disease (FMD) virus in aerosols to determine the minimal infective dose by the respiratory route. The aerosols used were produced either artificially by a spinning-top aerosol generator, in which case they were of homogeneous small particle size (less than 3 /-1m in diameter) or else they were derived naturally from infected pigs, in which case the particles were heterogeneous in size. Two strains of FMD virus were used: an 0. strain of UK origin and a SAT 2 strain from South Africa. The lowest doses which initiated infection were 12·5 TCID50 of 0. BFS virus and 25 TCID50 of SAT 2 virus, infectivity having been assayed in primary bovine thyroid cell cultures. Following exposure to low doses of virus (range 12 to 316 TCID50) 33 per cent of the cattle exposed to 0\ BFS virus and 27 per cent exposed to SAT 2 virus were infected but did not develop detectable vesicular lesions.
by using the models can be laid over maps of animal distribution to identify the herds at greatest risk downwind of a source of airborne virus emission. Apart from the preliminary experiments of Eskildsen (1969), who demonstrated the infection of a heifer using an aerosol dose of 5 x 1()2 mouse ID50, there is no published information on the minimal dose of airborne FMD virus for cattle. To operate the numerical prediction models in the absence of such data it has hitherto been necessary arbitrarily to define the dose for cattle, the species most likely to be infected in the event of airborne FMD spread (Gloster et al 1982). Consequently the precision of the models may have been suboptimal. Experiments have been undertaken to rectify this possible deficiency and the results obtained are presented in this paper.
THE quantities of airborne foot-and-mouth disease (FMD) virus produced by different species of infected animals and the various factors which can influence airborne virus survival and dispersion have been reviewed by Donaldson (1983) and Gloster et al (1982). Based on the results obtained in aerobiological and epidemiological investigations numerical models have been developed and successfully used for analysing the likelihood of airborne spread of FMD during outbreaks of the disease (Gloster et al 1981, Donaldson et aI1982). Two numerical models are available: a long-range model which is manual and can be used to analyse the risk of airborne spread from one coastal region to another across a sea passage; and a short-range model which is computer-based and can be used to examine the risk of spread over land for distances up to 10 km from a source. The dispersion plumes of airborne virus obtained
Twenty-eight calves weighing 109 to 166 kg and five calves weighing 43 to 46 kg were used. They were mainly crossbred Friesians but a few were crossbred AberdeenAngus or crossbred Hereford. The pigs used as sources of natural FMD virus aerosols were Large White cross Landrace weighing 20 to 25 kg.
~
Materials and methods
Animals
Virus strains
'
Two strains of FMD virus were used; strain 0, BFS 1860 originally isolated from cattle in the UK during the 1967 to 1968 (Oswestry) epidemic and strain SAT 2 SAR 3179, also of cattle origin, and kindly supplied by Dr G. R. Thomson, Foot-and-Mouth Disease Laboratory, Veterinary Research Institute, Onderstepoort, Republic of South Africa. The 0, BFS strain had been passaged three times in bovine tongue epithelial tissue and stored at - 20°C as a clarified suspension in 50:50 glycerol-phosphate
339
340
A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
buffer. High titre stock suspensions of virus were in BHK-21 cells, concentrated and partly purified as described by Ferris et al (1984) and stored at -70°C, Spray suspensions for aerosol generation were prepared by diluting concentrated virus in 0'1 per cent (w/v) nutrient broth (Oxoid) in distilled water adjusted to pH 7'4. By varying the dilution ?f vi~u~ i~ spray solutions the required range of infectivity In aerosols was obtained. The SAT virus strain was received as second passage cattle tongue epithelium. A I: 10 (w/v) stock preparation in 10 per cent nutrient broth was made, clarified by centrifugation, subdivided into I . 5 ml samples and stored at -700C, pre~~red
Sources oj FMD virus aerosols Two types of aerosol were used: artificial aerosols of 0 1 BFS virus generated by a May spinning-top apparatus modified according to Mitchell and Stone (1982); and natural aerosols produced by pigs infected with the SAT 2 virus. The latter aerosols are known to contain virus in association with heterogeneouslysized particles (Sellers and Parker 1969, Donaldson et al 1981) whereas the former consisted of homogeneous small particles.
Production oj natural aerosols . For each experiment four pigs were infected by Intradermal inoculation of the heel bulbs of a forefoot(Burrows 1966b) with stock SAT 2 virus diluted 1 10- in Eagle's medium. Each pig received about 5 0'3 ml of inoculum; a dose of around 10 "3 TCID50. Two days later, when most pigs in the group had generalised vesicular lesions, two were selected and placed in a 610 litre chamber (Gibson and Donaldson 1986) in the corridor of a high security animal isol~tion unit leading to the incinerator, that is, the section of the isolation compound with the highest negative air pressure. The pigs, being acutely ill, generally lay down on the floor of the chamber for the duration of the aerosol exposure section of the experiment. Before any calves were brought into the corridor for aerosol exposure the outside of the chamber, the corridor and the protective clothing, hands and boots of personnel were all thoroughly disinfected.
Production oj artificial aerosols With the exception of the May spinning-top and support column which were kindly supplied by the ......
Manometer point
Compound air duct )
2
2
d
t
Filter and iris diaphragm valve
Perspex cover Spinning<-l:.::."o,,-p-'-H--lI-ll-" Air pressure regulator and gauge Air feed to spinning top
Air sampler
Flow Pressure meter regulator Adjustable feet Steel base FIG 1: Diagram showing the components of the spinning top aerosol production and delivery apparatus (not drawn to scale)
i?
Z Z
b
Airborne FMD injection of cattle Centre for Applied Microbiological Research, Porton Down, Salisbury, and are commercially available from Research Engineers Ltd, London Nl 5RD, the remainder of the apparatus used for generating and delivering small-particle aerosols was made in the workshops of this institute. The apparatus includes the modifications recommended by Mitchell and Stone (1982) and a schematic diagram is provided in Fig 1. A supply of compressed air provided both the drive for the spinning-top and the dilution air flow to transport aerosol particles. A HEPA filter (Gelman Sciences) was sited in the compressed air line to remove particulate material. The air flow into the two air systems was controlled by pressure gauges and valves. The spent rotor drive air was removed through a high efficiencylow resistance filter (Microflow), suction being provided by a high volume variable-flow fan. Manometers were fitted to measure the dilution air and spent rotor drive air pressures. The system for delivering spray suspensions to the spinning-top consisted of a model 341 AF Sage motorised syringe (Arnold R. Horwell Ltd, London NW6 2BP), an allglass syringe, thin-bore plastic tubing and a 23 gauge hypodermic needle machine-ground to 1 cm in length with a squared-off end. A needle support mechanism with remote micrometer control was constructed to allow accurate needle height adjustments to be made when the top was spinning (Mitchell and Stone 1982). Testing and calibration of the equipment using spray suspension solutions containing various concentrations of nutrient broth in distilled water without virus was done in the laboratory with an aero,dynamic particle sizer (TSI Inc, Bristol Industrial and Research Associates) kindly loaned by Professor J. Webster and Dr C. Wathes, department of animal husbandry, University of Bristol. The conditions required to generate aerosols with an aerodynamic diameter of less than 3 !-1m were: spray suspension containing O' I per cent w/v nutrient broth; fluid delivery rate 1. 8 ml min - I; dilution air flow 7 psi and flow rate 70 litres min - I; pressure difference between dilution and exhaust air 4 mm of water. When the equipment had been calibrated in the laboratory it was transferred to the corridor of a high security animal isolation unit. Aerosols were delivered through a 32 mm diameter tube to an animal exposure port and thence along' 15 cm diameter vent ducting to the exit filter in the ceiling of the corridor of the isolation unit. To avoid suction effects due to the exhaust air system of the building, the vent ducting of the aerosol apparatus was not directly attached to the exhaust air filter but ended just underneath the filter housing. A by-pass tube was fitted across the animal exposure port so that a calf could be connected to the exposure port without being immediately exposed to virus aerosol.
341
Exposure oj calves to natural aerosols The apparatus used was the same as that described in a previous paper (Gibson and Donaldson 1986). Each calf was taken individually, connected via the exposure port and allowed to breath the air flowing from the chamber containing two pigs acutely infected with SAT 2 virus. The air flow in the ducting had previously been set at 0·5 m s -I by adjusting the iris diaphragm near the exit filter, readings being measured with an electronic anemometer. The air flow in the ducting was unidirectional, towards the exit filter. The exposure period for each calf was either five or 10minutes. During exposure the respiration rate of each calf was counted on each of four occasions and from these a mean respiration rate per minute was established. After the exposure of each animal it was placed in a separate isolation room to avoid cross infection. Each calf was examined daily for 14 days for clinical signs of FMD. Three experiments were carried out using five calves on each occasion.
Exposure oj calves to artificial aerosols Each calf was taken singly and connected via the exposure port to the apparatus illustrated in Fig 1. The bypass tube across the exposure tube was left open until the calf was breathing normally. Exposure was then begun. The period of exposure was 1, I . 5 or 2 minutes. During exposure all the respirations of a calf were counted. Following exposure each calf was housed separately and examined daily as described for calves exposed to natural aerosols. Three experiments were carried out using six calves in each experiment.
Air sampling Samples of artificial and natural virus aerosols were collected via tubing connected to the exposure port of the respective pieces of apparatus (Fig 1, Gibson and Donaldson 1986). The particle size association of infectious virus in the two types of aerosol was determined by collecting aerosol samples with a threestage liquid impinger (May 1966) operating at a sampling rate of 55 litres min - I for two to five minutes. Aerosol samples were collected just before and after a series of calves had been exposed to virus. Throughout the time each calf was being exposed a sample of the virus aerosol was collected with a Porton raised all-glass impinger (May and Harper 1957) attached to the exposure port. The sampling rate of each all-glass impinger, previously calibrated, was within the range 11 to 13 litres min -I. During the experiments with artificial virus aerosols where the exposure periods were very short, air sampling was extended beyond the time of exposure in order to
342
A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
TABLE 1: Doses of FMD virus, strain 0, BFS1860 in artificial aerosols to which calves were exposed
Weight (kg)
Respiration rate (min- 11
Exposure time (minI
Tidal volume (litre)
Air sampled" (litre I
Aerosol concentrationt (TCID50 litre." 1I
Dose'" (log,o TCID50)
PJ23 PJ24 PJ25 PJ26 PJ27 PJ28
112 118 124 109 121 127
24·0 17·0 23·0 16·0 22·0 18·0
1·0 1-5 1-5 1·5 1·0 1·0
1·17 1·30 1·37 1·17 1·30 1·37
28·1 33·1 47·3 28·1 28·6 24·7
5587·3 5475·1 503·1 489·5 368·9 589·3
5·2 5·2 4·4 4·1 4·0 4·2
PJ17 PJ18 PJ19 PJ20 PJ21 PJ22
143 158 118 162 166 154
15·0 18·0 18·0 14·0 18·0 15·0
2·0 1-5 2'0 1-5 1·0 1·0
1-63 1·83 1·30 1·83 1·90 1·77
48·9 49·4 48·8 38·4 34·2 26·5
9·1 5·1 6·2 2·1 2·3 0·9
2·6 2·4 2'5 1·9 1·9 1·4
PL18 PL19 PK78 PK79 PK80 PK81
45 48 43 46 112 46
12·0 20·0 28·0 25·0 20·0 24·0
1·0 1·0 1·0 1·0 1·0 1·0
0·32 0·32 0·25 0·32 1·17 0·32
3·8 6·4 7·0 8·0 23·4 7·6
Jo7
1·1 1·6 1·2 1·4 2·4
Animal number
6·9 2·1
Jo4
10·6 3·4
1-4
" Total volume of air sampled by animal during exposure = tidal volume (VI x respiration rate (rl x exposure time (t) t Infectivity recovered in air samplel Air flow of sampler x duration of air sampling = i :j: Vx r x t x i
ensure the recovery of virus. Sampling was continued until the syringe delivering the spray suspension was almost empty, a total time of from one minute 47 seconds to two minutes 40 seconds. After each series of calves had been exposed to either natural or artificial aerosol the air in the corridor was sampled for five minutes using a glass cyclone sampler (Errington and Powell 1969)operating at 700 litres min - , to test for background airborne virus. Aerosol collecting fluid The fluid used in the three-stage and cyclone air samplers was Eagle's medium containing 20 mM HEPES buffer and double-strength antibiotics, adjusted to pH 7· 2. The same fluid was used in the all-glass (porton) impingers with added 0'1 per cent silicone MS emulsion (Hopkin and Williams) to prevent foaming.
described by Hamblin et al (1986). An additional sample of serum for antibody assay was also taken about one week later from any animal in which evidence of infection was not found during the 14 days of clinical observation and testing. Around three weeks after exposure to virus, samples of oesophageal-pharyngeal (O-P) fluid were collected from all of the calves except numbers PJ 17 to 28. The method used was as described by Burrows (1966a). Samples were diluted in an equal volume of Eagle's medium containing 20 mM HEPES and doublestrength antibiotics, pH 7· 2, at 4°C, and inoculated into cell cultures within two hours after collection. Viral assay
Samples of heparinised blood and O-P fluid were tested for the presence' of virus by inoculating monolayer cultures of primary bovine thyroid cells (BTY) (Snowdon 1966)in roller tubes. The quantity of virus in air samples was determined by inoculating serial lO-fold dilutions into BTY cells as above. Between five Samples from animals and 10 tubes were used for each dilution. The speciBefore exposure to virus and daily thereafter for 14 ficity of cytopathic effect was confirmed byeither days, an heparinised and a non-heparinised blood complement fixation or ELISA. sample was taken from each animal using Vacutainer tubes (Becton Dickinson). Samples (1' 8 ml) of each Calculation of exposure dose whole blood sample were stored at -70°C and tested The relationship of bodyweight to respiratory tidal later for the presence of virus. Similar volumes of serum separated from each of the non-heparinised volume for cattle has been documented by Hales and samples were stored at - 20°C before testing by ELISA Findlay (1968). By plotting their data a correlation for the presence of antibodies to type 0, FMD virus as coefficient of the slope of 0'95 was obtained. This
343
Airborne FMD injection oj cattle TABLE 2: Response of calvas exposed to artificial aerosols of FMO virus. strain 0, BFS 1860
Earliest viraemia (l0910 TCI050) 3" 4 5 6 Dose
Animal number
PJ23 PJ24 PJ25 PJ26 PJ27 PJ28
5·2 5·2 4·4 4·1 4·0 4·2
PJ17 PJ18 PJ19 PJ20 PJ21 PJ22
2·6 2·4 2·5 1·9 1·9 1·4
pL18 PL19 PK78 PK79 PK80 PK81
1·1 1·6 1·2 1·4 2·4 1-4
+ +
7
8
Earliest lesions 5 6 7 8
+ + +
+
+ + +
9
10 6
+ + +
+
Earliest sero-conv 7 8 9 10
+
+
+ +
+
+ + +
+ + + +
+ +
+
fluid
NO NO NO NO NO NO
+ +
o-pt
+
+
+
NO NO NO NO NO NO
+
+
+ +
Infected
+ + + + + + + + + + + + +* +* -* -* +* +*
" t
Number of days after exposure to virus Oesophageal-pharyngeal * Specific antibody in an ELISA in serum taken at 14 days after exposure to virus. Serum samples not collected on days 1 to 13 NO Not examined
slope was taken as a standard and the tidal volume of each calf used in the experiments was estimated by reference to it. The infectivity dose (D) to which each calf was exposed was calculated from the equation: D=Vxrxtxi where
v = tidal
volume (litres) r = respiratory rate (min - ') t = exposure time (min) infectivity recovered in air sample (TC1D50 1- ') i = ------;--'--;----:--:----:-:----'-,---,:'-:---duration of air sampling (min) x air flow of sampler
D was expressed as 10gIO TC1D50. Results
Particle size association of airborne virus .. Above 85 per cent of the total airborne virus recovered from artificial aerosols was trapped in the bottom stage of the three-stage impinger and 15 per cent or less in the middle stage. Thus artificial aerosols contained particles which were predominantly less than 3 ~m in diameter, with a small proportion in the 3 to 6 ~m diameter size range (May 1966). Samples of natural aerosols yielded similar quantities of virus in all three stages of the impinger, demonstrating that in these aerosols virus was equally
associated with particles in the size ranges under 3, 3 to 6 and over 6 ~m diameter.
Air sampling with cyclone Virus was not recovered in any of the cyclone samples showing that any background virus which might have been present was below detectable con~ centration.
Response oj calves to artificial aerosols Calculation of the doses of artificial virus aerosol to which the calves were exposed showed that six calves (PJ23 to 28) received high exposure doses: within the infectivity range IOgI04 · 0- 5 . 2 TC1D50. The remaining 12 calves received low doses: range IOglO l " - 2' 6 TCID50 (Table I). All of the animals exposed to high doses were infected although one (PJ28) developed neither a detectable viraemia nor vesicular lesions (Table 2). This animal did, however, have a circulating antibody response from day 7 after exposure onwards. Following low dose exposure 10 of 12 calves (83' 3 per cent) were infected although in four cases (33' 3 per cent) signs of disease were inapparent. The lowest dose which initiated infection was 10gIO'" (12'5) K1D50. Five of the 18 calves (PJ28, PJI7, PJ18, PJI9 and PK80) did not have a detectable viraemia but seroconverted (Table 2). These animals were considered to have had a localised respiratory tract infection. Calf pL19 did not, however, have detectable virus in the single G-P fluid sample collected.
344
A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
TABLE 3: Doses of FMD virus. strain SAT 2 SAR 3/79 in natural aerosols to which calves were exposed
Weight (kg)
Respiration rate (min- 1)
Exposure time (min)
Tidal volume (litre)
Air sampled' (litre)
Aerosol concentrationt (TCID50 Iitre- 1)
TCID50)
po53 P054 P055 po56 po57
137 127 150 146 137
16·0 14·0 22·5 16·6 14·3
10·0 10·0 10·0 10·0 10·0
1·50 1·37 1·70 1·63 1·50
240·0 191·8 382·5 270·6 214·5
0·33 0·23 0·21 0·36 0·21
1·9 1·6 1·9 1·9 1-6
POO pol po2 po3 po4
132 140 146 136 150
11-4 21·2 17·0 16·6 20·2
10·0 10·0 10·0 10·0 10·0
1·44 1·57 1·63 1·50 1·70
164·2 332·8 277·1 249·0 343-4
0·15 0·15 0·15 0·21 0·33
1·4 1·7 1·6 1'7 2·1
PM54 PM55 PM56 PM57 PM58
137 118 134 134 127
42·0 21'0 17·0 13·0 21·5
5·0 5·0 5·0 5·0 5·0
1·50 1·30 1·50 1·50 1·37
315·0 136·5 127·5 97·5 147·3
0·85 0·41 0·41 0·46 0·42
2·4 1·7 1·7 1-6 1·8
Animal number
,
Dose:!: (l091O
Total volume of air sampled by animal during exposure = tidal volume (V) x respiration rate (r) x exposure time (t) t Infectivity collected in air sample/ Air flow of sampler x duration of air sampling = i :!: Vxrxtxi
TABLE 4: Response of calves to natural aerosols of FMD virus. strain SAT 2 SAR 3/79
Animal number
r-oss
Dose (log 10 TCID50)
po57
1·9 1·6 1·9 1·9 1·6
poO POl po2 po3 P04
1-4 1·7 1·6 1·7 2·1
PM54 PM55 PM56 PM57 PM58
2·4 1·7 1·7 1·6 1·8
p054 P055
ross
,
t
l'
+
Earliest viraemia 2 3
+
+
4
3
+ +
Earliest lesions 4 5 6
+ + +
+
+ + +
Earliest sero-conv
7
6
+ + +
7
+
+
+
8
9
10
O-P" fluid
Infected
+ + + + + +
+ + + + + +
+
+ +
+ +
+
+ + + +
'"
+ + +
Number of days after exposure to virus Oesophageal-pharyngeal
Response of calves to natural aerosols The calculated doses used to expose calves to natural aerosols were all low and within the infectivity range loglO l ' 4- 2-4 TCID50 (Table 3). Following exposure 11 of 15 (73' 3 per cent) of the calves were infected although of these four animals (26' 7 per cent) did not develop vesicular lesions. Three animals (PM 54, 57, 58) which seroconverted did not have a detectable viraemia. Animal po3 was considered to have been infected since it had a viraemia of two days
duration. It did not, however, have a detectable circulating antibody response. Virus was not isolated from the o-p fluid of this animal or from the three others which were considered to have been subclinically infected. Log 101' 4 (25) TCID50 was the lowest dose which produced infection. The incubation periods for calves exposed to natural virus aerosols (three to seven days) were in most cases shorter than those for calves which developed disease following exposure to low doses of artificial aerosols (six to 10 days).
Airborne FMD infection of cattle Discussion
345
pharyngeal inoculation. In the present investigations The results confirm that the respiratory route is the it is hypothesised that, in the case of the seven calves most susceptible natural portal of entry by which which seroconverted without developing lesions, cattle can be infected by FMD virus. The only other virus replication was restricted to sites in or associated route which approaches the respiratory tract in sensi- with the respiratory tract because the slower pathotivity is artificial, that is, intradermal inoculation of genesis of infection provided sufficient tiine for the activation of defence mechanisms which then conthe tongue (Sellers 1971). The minimum estimated dose of airborne FMD virus fined infection to that region. obtained for calves is close to that reported for sheep (Gibson and Donaldson 1986). The mathematical Acknowledgements models which have been developed for analysing the We are indebted to Mr I. S. Caie and Mr D. W. airborne spread of FMD virus (Gloster et al 1981, Donaldson et al 1982) have now been altered to take Richards, department of engineering and maintenance, A VRI, for the design and construction of these findings into account (Donaldson et aI1987). The aerosol concentrations used in the present aerosol generating equipment. Professor A. J. F. study were relatively high and so only short exposure Webster, Dr C. Wathes and Dr C. D. Jones, departperiods were required. During airborne transmission ment of animal husbandry, School of Veterinary under field conditions much lower concentrations are Science, University of Bristol, are thanked for the likely in virus plumes. The exposure of an animal for loan of the aerodynamic particle sizer. Dr J. P. several hours might, therefore, be necessary before it Mitchell, Atomic Energy Establishment, Winfrith, accumulates an infective dose within its respiratory Dorchester, and Dr C. D. Jones are thanked for tract. Normal respiratory clearance mechanisms will valuable advice about the construction of aerosol probably exert a greater influence under such equipment. The assistance of Mr N. P. Ferris, AVRI, in the testing of oesophageal-pharyngeal samples and extended exposure periods. for confirming the specificity of virus isolates is The slower onset of disease in the majority of calves exposed to artificial compared to natural aerosols (six gratefully acknowledged. to 10 versus three to seven days) suggests that the pathogenesis of disease after artificial aerosol exposure was not typical. Virus in artificial aerosols, References being predominantly associated with small particles, BURROWS. R. (1966a) Journal of Hygiene, Cambridge 64,81-90 would have lodged mainly in lower respiratory areas BURROWS, R. (I 966b) Journal ofHygiene, Cambridge 64, 419-429 whereas that in natural aerosols would have been BURROWS, R. (1968) Veterinary Record 82,387-388 deposited mainly in the airways of the head and ne~k BURROWS, R., MANN, J. A., GARLAND, A. J. M., GREIG, A. & GOODRIDGE, D. (1981) Journal of Comparative Pathology region (Hatch and Gross 1964). Eskildsen (1969) and 91,599-609 Sutmoller and McVicar (1976) demonstrated under BURROWS, R., MANN, J. A., GREIG, A., CHAPMAN, W. G. experimental conditions that infection in cattle can be & GOODRIDGE, D. (1971) Journal of Hygiene, Cambridge 69 307-321 ' initiated via the lungs but Burrows et al (1981) concluded that the usual route of primary invasion in DONALDSON, A. I. (1983) Philosophical Transactions of the Royal Society of London 8302, 529-534 cattle is via the mucosae and lymphoid tissues of the DONALDSON, A. I., GARLAND, A. J. M., FERRIS, N. P. & pharynx. FMD virus has a strong predilection for the COLLEN, T. (1981) British Veterinary Journal 137, 300-307 pharyngeal area of ruminants (Burrows 1968, DONALDSON, A. I., GLOSTER, J., HARVEY, L. D. J. & DEANS" D. H. (1982) Veterinary Record 110, 53-57 Burrows et al 1971, 1981, McVicar et al 1971) and airborne virus deposited initially in the lungs could D?NALDSON, A. I., LEE, M. & GIBSON, C. F. (1987) Advances In Aerobiology, Eds G. Boehm and R. M. Leuschnere. Birkhauser subsequently reach the pharyngeal region by upward Verlag, Basel. Proceedings of the Third International Aerotransportation on the mucociliary clearance system or biology Conference, Basel, Switzerland, August 6 to 9 1986. pp 351-355 alternatively by the bloodstream after traversing alveoli. By either of these secondary routes there !;:RRING!ON, F. P. & POWELL, E. O. (1969) Journal ofHygiene, Cambridge 67, 387-399 would be some clearance of infectivity. The longer ESKILDSEN, M. K. (1969) Nordisk Veterinaermedicin 21, 86-91 incubation periods found following artificial aerosol FERRIS, N. P., DONALDSON, A. I., BARNETT, I. T. R. & exposure may thus be a reflection of the longer time OSBORNE, R. W. (1984) Revue Scientifique et Technique de I'OIE 3,339-350.' taken by virus to reach threshold infectivity levels in GIBSON, C. F. ~ DONALDSON, A. I. (1986) Research in more favoured sites of replication. Veterinary Science 41,45-49 The production of subclinical infection in some GLOSTER,. J., B'LACKALL, R. M., SELLERS, R. F. & calves, particularly with low dose exposure, is in DONALDSON, A. I. (1981) Veterinary Record 108, 370-374 agreement with the findings of Sutmoller et al (1968) GLOSTER, J., SELLERS, R. F. & DONALDSON, A. I. (1982) Veterinary Record 110, 47-52 w~~ reported inapparent infection in cattle given HALES, J. R. S. & FINDLAY, J. D. (1968) Respiration Physiology minimal amounts of FMD virus by intranasal or intra4,333-352
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A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
HAMBLIN, c., BARNETT, I. T. R. & HEDGER, R. S. (1986) Journal of Immunological Methods 93,115-121 HATCH, T. F. & GROSS, P. (1964) Pulmonary Deposition and Retention of Inhaled Aerosols. London, Academic Press. pp 45-68 MAY, K. R. (1966) Bacteriological Reviews 30,559-570 MAY, K. R. & HARPER, G. J. (1957) British Journal of Industrial Medicine 14, 287-297 MITCHELL, J. P. & STONE, R. L. (1982) Journal of Physics 15, 565-567 McVICAR, J. W., GRAVES, J. H. & SUTMOLLER, P. (1971) United States Animal Health Association Proceedings 74, 230-234
SELLERS, R. F. (1971) Veterinary Bulletin 41, 431-439 SELLERS, R. F. & PARKER, J. (1969) Journal o}\ Hygiene, Cambridge 67, 671-677 SNOWDON, W. A. (1966) Nature 210,1079-1080 SUTMOLLER, P. & McVICAR, J. W. (1976) Journal of Hygiene 77,235-243 SUTMOLLER, P., McVICAR, J. W. & COTTRAL, G. E. (1968) Archiv fur die gesamte Virusforschung 23, 227-235
Received November 19, 1986 Accepted December 18, 1986
Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with 01 and SAT 2 strains A. 1. DONALDSON, Animal Virus Research Institute, Pirbright, Woking, Surrey, GU240NF, C. F. GIBSON, May and Baker Ltd, Rainham Road South, Dagenham, Essex, RMIO 7XS, R. OLIVER, Central Animal Health Laboratory, Ministry of Agriculture and Fisheries, Wallaceville Animal Research Centre, Private Bag, Upper Hutt, New Zealand, C. HAMBLIN, R. P. KITCHING, Animal Virus Research Institute, Pirbright, Woking, Surrey, GU240NF
Equipment has been constructed and methods developed for exposing individual cattle to two strains of foot-and-mouth disease (FMD) virus in aerosols to determine the minimal infective dose by the respiratory route. The aerosols used were produced either artificially by a spinning-top aerosol generator, in which case they were of homogeneous small particle size (less than 3 /-1m in diameter) or else they were derived naturally from infected pigs, in which case the particles were heterogeneous in size. Two strains of FMD virus were used: an 0. strain of UK origin and a SAT 2 strain from South Africa. The lowest doses which initiated infection were 12·5 TCID50 of 0. BFS virus and 25 TCID50 of SAT 2 virus, infectivity having been assayed in primary bovine thyroid cell cultures. Following exposure to low doses of virus (range 12 to 316 TCID50) 33 per cent of the cattle exposed to 0\ BFS virus and 27 per cent exposed to SAT 2 virus were infected but did not develop detectable vesicular lesions.
by using the models can be laid over maps of animal distribution to identify the herds at greatest risk downwind of a source of airborne virus emission. Apart from the preliminary experiments of Eskildsen (1969), who demonstrated the infection of a heifer using an aerosol dose of 5 x 1()2 mouse ID50, there is no published information on the minimal dose of airborne FMD virus for cattle. To operate the numerical prediction models in the absence of such data it has hitherto been necessary arbitrarily to define the dose for cattle, the species most likely to be infected in the event of airborne FMD spread (Gloster et al 1982). Consequently the precision of the models may have been suboptimal. Experiments have been undertaken to rectify this possible deficiency and the results obtained are presented in this paper.
THE quantities of airborne foot-and-mouth disease (FMD) virus produced by different species of infected animals and the various factors which can influence airborne virus survival and dispersion have been reviewed by Donaldson (1983) and Gloster et al (1982). Based on the results obtained in aerobiological and epidemiological investigations numerical models have been developed and successfully used for analysing the likelihood of airborne spread of FMD during outbreaks of the disease (Gloster et al 1981, Donaldson et aI1982). Two numerical models are available: a long-range model which is manual and can be used to analyse the risk of airborne spread from one coastal region to another across a sea passage; and a short-range model which is computer-based and can be used to examine the risk of spread over land for distances up to 10 km from a source. The dispersion plumes of airborne virus obtained
Twenty-eight calves weighing 109 to 166 kg and five calves weighing 43 to 46 kg were used. They were mainly crossbred Friesians but a few were crossbred AberdeenAngus or crossbred Hereford. The pigs used as sources of natural FMD virus aerosols were Large White cross Landrace weighing 20 to 25 kg.
~
Materials and methods
Animals
Virus strains
'
Two strains of FMD virus were used; strain 0, BFS 1860 originally isolated from cattle in the UK during the 1967 to 1968 (Oswestry) epidemic and strain SAT 2 SAR 3179, also of cattle origin, and kindly supplied by Dr G. R. Thomson, Foot-and-Mouth Disease Laboratory, Veterinary Research Institute, Onderstepoort, Republic of South Africa. The 0, BFS strain had been passaged three times in bovine tongue epithelial tissue and stored at - 20°C as a clarified suspension in 50:50 glycerol-phosphate
339
340
A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
buffer. High titre stock suspensions of virus were in BHK-21 cells, concentrated and partly purified as described by Ferris et al (1984) and stored at -70°C, Spray suspensions for aerosol generation were prepared by diluting concentrated virus in 0'1 per cent (w/v) nutrient broth (Oxoid) in distilled water adjusted to pH 7'4. By varying the dilution ?f vi~u~ i~ spray solutions the required range of infectivity In aerosols was obtained. The SAT virus strain was received as second passage cattle tongue epithelium. A I: 10 (w/v) stock preparation in 10 per cent nutrient broth was made, clarified by centrifugation, subdivided into I . 5 ml samples and stored at -700C, pre~~red
Sources oj FMD virus aerosols Two types of aerosol were used: artificial aerosols of 0 1 BFS virus generated by a May spinning-top apparatus modified according to Mitchell and Stone (1982); and natural aerosols produced by pigs infected with the SAT 2 virus. The latter aerosols are known to contain virus in association with heterogeneouslysized particles (Sellers and Parker 1969, Donaldson et al 1981) whereas the former consisted of homogeneous small particles.
Production oj natural aerosols . For each experiment four pigs were infected by Intradermal inoculation of the heel bulbs of a forefoot(Burrows 1966b) with stock SAT 2 virus diluted 1 10- in Eagle's medium. Each pig received about 5 0'3 ml of inoculum; a dose of around 10 "3 TCID50. Two days later, when most pigs in the group had generalised vesicular lesions, two were selected and placed in a 610 litre chamber (Gibson and Donaldson 1986) in the corridor of a high security animal isol~tion unit leading to the incinerator, that is, the section of the isolation compound with the highest negative air pressure. The pigs, being acutely ill, generally lay down on the floor of the chamber for the duration of the aerosol exposure section of the experiment. Before any calves were brought into the corridor for aerosol exposure the outside of the chamber, the corridor and the protective clothing, hands and boots of personnel were all thoroughly disinfected.
Production oj artificial aerosols With the exception of the May spinning-top and support column which were kindly supplied by the ......
Manometer point
Compound air duct )
2
2
d
t
Filter and iris diaphragm valve
Perspex cover Spinning<-l:.::."o,,-p-'-H--lI-ll-" Air pressure regulator and gauge Air feed to spinning top
Air sampler
Flow Pressure meter regulator Adjustable feet Steel base FIG 1: Diagram showing the components of the spinning top aerosol production and delivery apparatus (not drawn to scale)
i?
Z Z
b
Airborne FMD injection of cattle Centre for Applied Microbiological Research, Porton Down, Salisbury, and are commercially available from Research Engineers Ltd, London Nl 5RD, the remainder of the apparatus used for generating and delivering small-particle aerosols was made in the workshops of this institute. The apparatus includes the modifications recommended by Mitchell and Stone (1982) and a schematic diagram is provided in Fig 1. A supply of compressed air provided both the drive for the spinning-top and the dilution air flow to transport aerosol particles. A HEPA filter (Gelman Sciences) was sited in the compressed air line to remove particulate material. The air flow into the two air systems was controlled by pressure gauges and valves. The spent rotor drive air was removed through a high efficiencylow resistance filter (Microflow), suction being provided by a high volume variable-flow fan. Manometers were fitted to measure the dilution air and spent rotor drive air pressures. The system for delivering spray suspensions to the spinning-top consisted of a model 341 AF Sage motorised syringe (Arnold R. Horwell Ltd, London NW6 2BP), an allglass syringe, thin-bore plastic tubing and a 23 gauge hypodermic needle machine-ground to 1 cm in length with a squared-off end. A needle support mechanism with remote micrometer control was constructed to allow accurate needle height adjustments to be made when the top was spinning (Mitchell and Stone 1982). Testing and calibration of the equipment using spray suspension solutions containing various concentrations of nutrient broth in distilled water without virus was done in the laboratory with an aero,dynamic particle sizer (TSI Inc, Bristol Industrial and Research Associates) kindly loaned by Professor J. Webster and Dr C. Wathes, department of animal husbandry, University of Bristol. The conditions required to generate aerosols with an aerodynamic diameter of less than 3 !-1m were: spray suspension containing O' I per cent w/v nutrient broth; fluid delivery rate 1. 8 ml min - I; dilution air flow 7 psi and flow rate 70 litres min - I; pressure difference between dilution and exhaust air 4 mm of water. When the equipment had been calibrated in the laboratory it was transferred to the corridor of a high security animal isolation unit. Aerosols were delivered through a 32 mm diameter tube to an animal exposure port and thence along' 15 cm diameter vent ducting to the exit filter in the ceiling of the corridor of the isolation unit. To avoid suction effects due to the exhaust air system of the building, the vent ducting of the aerosol apparatus was not directly attached to the exhaust air filter but ended just underneath the filter housing. A by-pass tube was fitted across the animal exposure port so that a calf could be connected to the exposure port without being immediately exposed to virus aerosol.
341
Exposure oj calves to natural aerosols The apparatus used was the same as that described in a previous paper (Gibson and Donaldson 1986). Each calf was taken individually, connected via the exposure port and allowed to breath the air flowing from the chamber containing two pigs acutely infected with SAT 2 virus. The air flow in the ducting had previously been set at 0·5 m s -I by adjusting the iris diaphragm near the exit filter, readings being measured with an electronic anemometer. The air flow in the ducting was unidirectional, towards the exit filter. The exposure period for each calf was either five or 10minutes. During exposure the respiration rate of each calf was counted on each of four occasions and from these a mean respiration rate per minute was established. After the exposure of each animal it was placed in a separate isolation room to avoid cross infection. Each calf was examined daily for 14 days for clinical signs of FMD. Three experiments were carried out using five calves on each occasion.
Exposure oj calves to artificial aerosols Each calf was taken singly and connected via the exposure port to the apparatus illustrated in Fig 1. The bypass tube across the exposure tube was left open until the calf was breathing normally. Exposure was then begun. The period of exposure was 1, I . 5 or 2 minutes. During exposure all the respirations of a calf were counted. Following exposure each calf was housed separately and examined daily as described for calves exposed to natural aerosols. Three experiments were carried out using six calves in each experiment.
Air sampling Samples of artificial and natural virus aerosols were collected via tubing connected to the exposure port of the respective pieces of apparatus (Fig 1, Gibson and Donaldson 1986). The particle size association of infectious virus in the two types of aerosol was determined by collecting aerosol samples with a threestage liquid impinger (May 1966) operating at a sampling rate of 55 litres min - I for two to five minutes. Aerosol samples were collected just before and after a series of calves had been exposed to virus. Throughout the time each calf was being exposed a sample of the virus aerosol was collected with a Porton raised all-glass impinger (May and Harper 1957) attached to the exposure port. The sampling rate of each all-glass impinger, previously calibrated, was within the range 11 to 13 litres min -I. During the experiments with artificial virus aerosols where the exposure periods were very short, air sampling was extended beyond the time of exposure in order to
342
A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
TABLE 1: Doses of FMD virus, strain 0, BFS1860 in artificial aerosols to which calves were exposed
Weight (kg)
Respiration rate (min- 11
Exposure time (minI
Tidal volume (litre)
Air sampled" (litre I
Aerosol concentrationt (TCID50 litre." 1I
Dose'" (log,o TCID50)
PJ23 PJ24 PJ25 PJ26 PJ27 PJ28
112 118 124 109 121 127
24·0 17·0 23·0 16·0 22·0 18·0
1·0 1-5 1-5 1·5 1·0 1·0
1·17 1·30 1·37 1·17 1·30 1·37
28·1 33·1 47·3 28·1 28·6 24·7
5587·3 5475·1 503·1 489·5 368·9 589·3
5·2 5·2 4·4 4·1 4·0 4·2
PJ17 PJ18 PJ19 PJ20 PJ21 PJ22
143 158 118 162 166 154
15·0 18·0 18·0 14·0 18·0 15·0
2·0 1-5 2'0 1-5 1·0 1·0
1-63 1·83 1·30 1·83 1·90 1·77
48·9 49·4 48·8 38·4 34·2 26·5
9·1 5·1 6·2 2·1 2·3 0·9
2·6 2·4 2'5 1·9 1·9 1·4
PL18 PL19 PK78 PK79 PK80 PK81
45 48 43 46 112 46
12·0 20·0 28·0 25·0 20·0 24·0
1·0 1·0 1·0 1·0 1·0 1·0
0·32 0·32 0·25 0·32 1·17 0·32
3·8 6·4 7·0 8·0 23·4 7·6
Jo7
1·1 1·6 1·2 1·4 2·4
Animal number
6·9 2·1
Jo4
10·6 3·4
1-4
" Total volume of air sampled by animal during exposure = tidal volume (VI x respiration rate (rl x exposure time (t) t Infectivity recovered in air samplel Air flow of sampler x duration of air sampling = i :j: Vx r x t x i
ensure the recovery of virus. Sampling was continued until the syringe delivering the spray suspension was almost empty, a total time of from one minute 47 seconds to two minutes 40 seconds. After each series of calves had been exposed to either natural or artificial aerosol the air in the corridor was sampled for five minutes using a glass cyclone sampler (Errington and Powell 1969)operating at 700 litres min - , to test for background airborne virus. Aerosol collecting fluid The fluid used in the three-stage and cyclone air samplers was Eagle's medium containing 20 mM HEPES buffer and double-strength antibiotics, adjusted to pH 7· 2. The same fluid was used in the all-glass (porton) impingers with added 0'1 per cent silicone MS emulsion (Hopkin and Williams) to prevent foaming.
described by Hamblin et al (1986). An additional sample of serum for antibody assay was also taken about one week later from any animal in which evidence of infection was not found during the 14 days of clinical observation and testing. Around three weeks after exposure to virus, samples of oesophageal-pharyngeal (O-P) fluid were collected from all of the calves except numbers PJ 17 to 28. The method used was as described by Burrows (1966a). Samples were diluted in an equal volume of Eagle's medium containing 20 mM HEPES and doublestrength antibiotics, pH 7· 2, at 4°C, and inoculated into cell cultures within two hours after collection. Viral assay
Samples of heparinised blood and O-P fluid were tested for the presence' of virus by inoculating monolayer cultures of primary bovine thyroid cells (BTY) (Snowdon 1966)in roller tubes. The quantity of virus in air samples was determined by inoculating serial lO-fold dilutions into BTY cells as above. Between five Samples from animals and 10 tubes were used for each dilution. The speciBefore exposure to virus and daily thereafter for 14 ficity of cytopathic effect was confirmed byeither days, an heparinised and a non-heparinised blood complement fixation or ELISA. sample was taken from each animal using Vacutainer tubes (Becton Dickinson). Samples (1' 8 ml) of each Calculation of exposure dose whole blood sample were stored at -70°C and tested The relationship of bodyweight to respiratory tidal later for the presence of virus. Similar volumes of serum separated from each of the non-heparinised volume for cattle has been documented by Hales and samples were stored at - 20°C before testing by ELISA Findlay (1968). By plotting their data a correlation for the presence of antibodies to type 0, FMD virus as coefficient of the slope of 0'95 was obtained. This
343
Airborne FMD injection oj cattle TABLE 2: Response of calvas exposed to artificial aerosols of FMO virus. strain 0, BFS 1860
Earliest viraemia (l0910 TCI050) 3" 4 5 6 Dose
Animal number
PJ23 PJ24 PJ25 PJ26 PJ27 PJ28
5·2 5·2 4·4 4·1 4·0 4·2
PJ17 PJ18 PJ19 PJ20 PJ21 PJ22
2·6 2·4 2·5 1·9 1·9 1·4
pL18 PL19 PK78 PK79 PK80 PK81
1·1 1·6 1·2 1·4 2·4 1-4
+ +
7
8
Earliest lesions 5 6 7 8
+ + +
+
+ + +
9
10 6
+ + +
+
Earliest sero-conv 7 8 9 10
+
+
+ +
+
+ + +
+ + + +
+ +
+
fluid
NO NO NO NO NO NO
+ +
o-pt
+
+
+
NO NO NO NO NO NO
+
+
+ +
Infected
+ + + + + + + + + + + + +* +* -* -* +* +*
" t
Number of days after exposure to virus Oesophageal-pharyngeal * Specific antibody in an ELISA in serum taken at 14 days after exposure to virus. Serum samples not collected on days 1 to 13 NO Not examined
slope was taken as a standard and the tidal volume of each calf used in the experiments was estimated by reference to it. The infectivity dose (D) to which each calf was exposed was calculated from the equation: D=Vxrxtxi where
v = tidal
volume (litres) r = respiratory rate (min - ') t = exposure time (min) infectivity recovered in air sample (TC1D50 1- ') i = ------;--'--;----:--:----:-:----'-,---,:'-:---duration of air sampling (min) x air flow of sampler
D was expressed as 10gIO TC1D50. Results
Particle size association of airborne virus .. Above 85 per cent of the total airborne virus recovered from artificial aerosols was trapped in the bottom stage of the three-stage impinger and 15 per cent or less in the middle stage. Thus artificial aerosols contained particles which were predominantly less than 3 ~m in diameter, with a small proportion in the 3 to 6 ~m diameter size range (May 1966). Samples of natural aerosols yielded similar quantities of virus in all three stages of the impinger, demonstrating that in these aerosols virus was equally
associated with particles in the size ranges under 3, 3 to 6 and over 6 ~m diameter.
Air sampling with cyclone Virus was not recovered in any of the cyclone samples showing that any background virus which might have been present was below detectable con~ centration.
Response oj calves to artificial aerosols Calculation of the doses of artificial virus aerosol to which the calves were exposed showed that six calves (PJ23 to 28) received high exposure doses: within the infectivity range IOgI04 · 0- 5 . 2 TC1D50. The remaining 12 calves received low doses: range IOglO l " - 2' 6 TCID50 (Table I). All of the animals exposed to high doses were infected although one (PJ28) developed neither a detectable viraemia nor vesicular lesions (Table 2). This animal did, however, have a circulating antibody response from day 7 after exposure onwards. Following low dose exposure 10 of 12 calves (83' 3 per cent) were infected although in four cases (33' 3 per cent) signs of disease were inapparent. The lowest dose which initiated infection was 10gIO'" (12'5) K1D50. Five of the 18 calves (PJ28, PJI7, PJ18, PJI9 and PK80) did not have a detectable viraemia but seroconverted (Table 2). These animals were considered to have had a localised respiratory tract infection. Calf pL19 did not, however, have detectable virus in the single G-P fluid sample collected.
344
A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
TABLE 3: Doses of FMD virus. strain SAT 2 SAR 3/79 in natural aerosols to which calves were exposed
Weight (kg)
Respiration rate (min- 1)
Exposure time (min)
Tidal volume (litre)
Air sampled' (litre)
Aerosol concentrationt (TCID50 Iitre- 1)
TCID50)
po53 P054 P055 po56 po57
137 127 150 146 137
16·0 14·0 22·5 16·6 14·3
10·0 10·0 10·0 10·0 10·0
1·50 1·37 1·70 1·63 1·50
240·0 191·8 382·5 270·6 214·5
0·33 0·23 0·21 0·36 0·21
1·9 1·6 1·9 1·9 1-6
POO pol po2 po3 po4
132 140 146 136 150
11-4 21·2 17·0 16·6 20·2
10·0 10·0 10·0 10·0 10·0
1·44 1·57 1·63 1·50 1·70
164·2 332·8 277·1 249·0 343-4
0·15 0·15 0·15 0·21 0·33
1·4 1·7 1·6 1'7 2·1
PM54 PM55 PM56 PM57 PM58
137 118 134 134 127
42·0 21'0 17·0 13·0 21·5
5·0 5·0 5·0 5·0 5·0
1·50 1·30 1·50 1·50 1·37
315·0 136·5 127·5 97·5 147·3
0·85 0·41 0·41 0·46 0·42
2·4 1·7 1·7 1-6 1·8
Animal number
,
Dose:!: (l091O
Total volume of air sampled by animal during exposure = tidal volume (V) x respiration rate (r) x exposure time (t) t Infectivity collected in air sample/ Air flow of sampler x duration of air sampling = i :!: Vxrxtxi
TABLE 4: Response of calves to natural aerosols of FMD virus. strain SAT 2 SAR 3/79
Animal number
r-oss
Dose (log 10 TCID50)
po57
1·9 1·6 1·9 1·9 1·6
poO POl po2 po3 P04
1-4 1·7 1·6 1·7 2·1
PM54 PM55 PM56 PM57 PM58
2·4 1·7 1·7 1·6 1·8
p054 P055
ross
,
t
l'
+
Earliest viraemia 2 3
+
+
4
3
+ +
Earliest lesions 4 5 6
+ + +
+
+ + +
Earliest sero-conv
7
6
+ + +
7
+
+
+
8
9
10
O-P" fluid
Infected
+ + + + + +
+ + + + + +
+
+ +
+ +
+
+ + + +
'"
+ + +
Number of days after exposure to virus Oesophageal-pharyngeal
Response of calves to natural aerosols The calculated doses used to expose calves to natural aerosols were all low and within the infectivity range loglO l ' 4- 2-4 TCID50 (Table 3). Following exposure 11 of 15 (73' 3 per cent) of the calves were infected although of these four animals (26' 7 per cent) did not develop vesicular lesions. Three animals (PM 54, 57, 58) which seroconverted did not have a detectable viraemia. Animal po3 was considered to have been infected since it had a viraemia of two days
duration. It did not, however, have a detectable circulating antibody response. Virus was not isolated from the o-p fluid of this animal or from the three others which were considered to have been subclinically infected. Log 101' 4 (25) TCID50 was the lowest dose which produced infection. The incubation periods for calves exposed to natural virus aerosols (three to seven days) were in most cases shorter than those for calves which developed disease following exposure to low doses of artificial aerosols (six to 10 days).
Airborne FMD infection of cattle Discussion
345
pharyngeal inoculation. In the present investigations The results confirm that the respiratory route is the it is hypothesised that, in the case of the seven calves most susceptible natural portal of entry by which which seroconverted without developing lesions, cattle can be infected by FMD virus. The only other virus replication was restricted to sites in or associated route which approaches the respiratory tract in sensi- with the respiratory tract because the slower pathotivity is artificial, that is, intradermal inoculation of genesis of infection provided sufficient tiine for the activation of defence mechanisms which then conthe tongue (Sellers 1971). The minimum estimated dose of airborne FMD virus fined infection to that region. obtained for calves is close to that reported for sheep (Gibson and Donaldson 1986). The mathematical Acknowledgements models which have been developed for analysing the We are indebted to Mr I. S. Caie and Mr D. W. airborne spread of FMD virus (Gloster et al 1981, Donaldson et al 1982) have now been altered to take Richards, department of engineering and maintenance, A VRI, for the design and construction of these findings into account (Donaldson et aI1987). The aerosol concentrations used in the present aerosol generating equipment. Professor A. J. F. study were relatively high and so only short exposure Webster, Dr C. Wathes and Dr C. D. Jones, departperiods were required. During airborne transmission ment of animal husbandry, School of Veterinary under field conditions much lower concentrations are Science, University of Bristol, are thanked for the likely in virus plumes. The exposure of an animal for loan of the aerodynamic particle sizer. Dr J. P. several hours might, therefore, be necessary before it Mitchell, Atomic Energy Establishment, Winfrith, accumulates an infective dose within its respiratory Dorchester, and Dr C. D. Jones are thanked for tract. Normal respiratory clearance mechanisms will valuable advice about the construction of aerosol probably exert a greater influence under such equipment. The assistance of Mr N. P. Ferris, AVRI, in the testing of oesophageal-pharyngeal samples and extended exposure periods. for confirming the specificity of virus isolates is The slower onset of disease in the majority of calves exposed to artificial compared to natural aerosols (six gratefully acknowledged. to 10 versus three to seven days) suggests that the pathogenesis of disease after artificial aerosol exposure was not typical. Virus in artificial aerosols, References being predominantly associated with small particles, BURROWS. R. (1966a) Journal of Hygiene, Cambridge 64,81-90 would have lodged mainly in lower respiratory areas BURROWS, R. (I 966b) Journal ofHygiene, Cambridge 64, 419-429 whereas that in natural aerosols would have been BURROWS, R. (1968) Veterinary Record 82,387-388 deposited mainly in the airways of the head and ne~k BURROWS, R., MANN, J. A., GARLAND, A. J. M., GREIG, A. & GOODRIDGE, D. (1981) Journal of Comparative Pathology region (Hatch and Gross 1964). Eskildsen (1969) and 91,599-609 Sutmoller and McVicar (1976) demonstrated under BURROWS, R., MANN, J. A., GREIG, A., CHAPMAN, W. G. experimental conditions that infection in cattle can be & GOODRIDGE, D. (1971) Journal of Hygiene, Cambridge 69 307-321 ' initiated via the lungs but Burrows et al (1981) concluded that the usual route of primary invasion in DONALDSON, A. I. (1983) Philosophical Transactions of the Royal Society of London 8302, 529-534 cattle is via the mucosae and lymphoid tissues of the DONALDSON, A. I., GARLAND, A. J. M., FERRIS, N. P. & pharynx. FMD virus has a strong predilection for the COLLEN, T. (1981) British Veterinary Journal 137, 300-307 pharyngeal area of ruminants (Burrows 1968, DONALDSON, A. I., GLOSTER, J., HARVEY, L. D. J. & DEANS" D. H. (1982) Veterinary Record 110, 53-57 Burrows et al 1971, 1981, McVicar et al 1971) and airborne virus deposited initially in the lungs could D?NALDSON, A. I., LEE, M. & GIBSON, C. F. (1987) Advances In Aerobiology, Eds G. Boehm and R. M. Leuschnere. Birkhauser subsequently reach the pharyngeal region by upward Verlag, Basel. Proceedings of the Third International Aerotransportation on the mucociliary clearance system or biology Conference, Basel, Switzerland, August 6 to 9 1986. pp 351-355 alternatively by the bloodstream after traversing alveoli. By either of these secondary routes there !;:RRING!ON, F. P. & POWELL, E. O. (1969) Journal ofHygiene, Cambridge 67, 387-399 would be some clearance of infectivity. The longer ESKILDSEN, M. K. (1969) Nordisk Veterinaermedicin 21, 86-91 incubation periods found following artificial aerosol FERRIS, N. P., DONALDSON, A. I., BARNETT, I. T. R. & exposure may thus be a reflection of the longer time OSBORNE, R. W. (1984) Revue Scientifique et Technique de I'OIE 3,339-350.' taken by virus to reach threshold infectivity levels in GIBSON, C. F. ~ DONALDSON, A. I. (1986) Research in more favoured sites of replication. Veterinary Science 41,45-49 The production of subclinical infection in some GLOSTER,. J., B'LACKALL, R. M., SELLERS, R. F. & calves, particularly with low dose exposure, is in DONALDSON, A. I. (1981) Veterinary Record 108, 370-374 agreement with the findings of Sutmoller et al (1968) GLOSTER, J., SELLERS, R. F. & DONALDSON, A. I. (1982) Veterinary Record 110, 47-52 w~~ reported inapparent infection in cattle given HALES, J. R. S. & FINDLAY, J. D. (1968) Respiration Physiology minimal amounts of FMD virus by intranasal or intra4,333-352
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A. I. Donaldson, C. F. Gibson, R. Oliver, C. Hamblin, R. P. Kitching
HAMBLIN, c., BARNETT, I. T. R. & HEDGER, R. S. (1986) Journal of Immunological Methods 93,115-121 HATCH, T. F. & GROSS, P. (1964) Pulmonary Deposition and Retention of Inhaled Aerosols. London, Academic Press. pp 45-68 MAY, K. R. (1966) Bacteriological Reviews 30,559-570 MAY, K. R. & HARPER, G. J. (1957) British Journal of Industrial Medicine 14, 287-297 MITCHELL, J. P. & STONE, R. L. (1982) Journal of Physics 15, 565-567 McVICAR, J. W., GRAVES, J. H. & SUTMOLLER, P. (1971) United States Animal Health Association Proceedings 74, 230-234
SELLERS, R. F. (1971) Veterinary Bulletin 41, 431-439 SELLERS, R. F. & PARKER, J. (1969) Journal o}\ Hygiene, Cambridge 67, 671-677 SNOWDON, W. A. (1966) Nature 210,1079-1080 SUTMOLLER, P. & McVICAR, J. W. (1976) Journal of Hygiene 77,235-243 SUTMOLLER, P., McVICAR, J. W. & COTTRAL, G. E. (1968) Archiv fur die gesamte Virusforschung 23, 227-235
Received November 19, 1986 Accepted December 18, 1986