Airborne infection with the virus of foot-and-mouth disease

.J.

COMP.

PATH.

1965.

VOL.

AIRBORNE

119

75.

INFECTION WITH THE VIRUS FOOT-AND-MOUTH DISEASE

OF

BY

N. Animal

ST.

G.

HYSLOP

Virus Research Institute,

Pitbright,

Surrey

INTRODUCTION

There can be little doubt that the principal mode of spread of foot-and-mouth disease (F.M.D.) is by either direct or indirect contact with infected animals or carcases, contaminated straw, fodder, etc. Nevertheless, a number of instances have occurred in the past in which it has been possible to infer that the virus has been disseminated locally in air currents and in this way has passed natural or artificial barriers. In addition to examples of local spread, it has been suggested that F.M.D. virus may be carried by the wind for long distances in the way that dust, smoke and pollen grains may travel for hundreds of miles in high altitude air currents. McLean (1938) indicated that physical adsorption onto fine particulate matter of this nature may be an important factor in the aerial spread of the disease.Whether or not F.M.D. virus would remain viable in transit for long periods is problematical and this might depend principally on the prevailing conditions of sunlight, temperature, humidity, etc. There are records of several instancesin which F.M.D. virus may have been carried for quite long distances by air currents. Thus, for example, Danish investigators have attributed outbreaks on the Islands of Denmark, during periods of strong S. or SW. winds, to airborne spread from Germany. However, it is impossibleto exclude human or other contacts, and particularly birds, between these islands and Germany, but it is especially interesting to note that the diseasespread only slowly by contact across the land frontier into the Jutland part of Denmark. Similarly, primary outbreaks have occurred in Southern Norway and Sweden, often on very isolated farms, at times when the diseasewas prevalent in Northern Denmark; direct contact between thesefarms and Denmark wasconsidered to be unlikely, though Jerlov (1940) recorded an instance in which infection was believed to have been introduced by a German fishing boat. Although reports from field sources tend to support the contention that F.M.D. occasionally may be a true airborne infection, experimental evidence is both scarce and conflicting. Thus, in an experiment during which the air in a building was deliberately contaminated with sterilized hay dust, Fogedby, Malmquist, Osteen and Johnson (1960) found that susceptiblecattle eventually became infected when placed in an air draught directed acrossinfected donor cattle maintained at a distance of 10 metres. In this case it remains a matter of opinion whether the infection was airborne or dustborne. In an experiment of a somewhat similar nature Traub and Wittman (1957), who did not contaminate the atmosphere with dust, failed to infect calves and pi lets by exposing them to air drawn from a shed containing infected animals. SimiParly, Moosbrugger (1948) recorded that a calf kept in a building used for virus production did not become infected as a result of airborne virus, but was infected by contaminated hay. On the other hand, Hyslop (1957, 1963), using an apparatus designed for studies on contagious bovine pleuropneumonia, infected 2 steersby exposing them to an experimentally-generated aerosol of F.M.D. virus.

120

AIRBORNE

INFECTION

WITH

F.

AND

M.

VIRUS

In a recent paper (Hyslop, 1965) i t was shown that infected cattle secrete large amounts of virus in their saliva, and it is possible, theoretically, for a high proportion of droplets of the size which tend to remain in aerial suspension for long periods (< 12 microns diam.) to contain viable virus particles. The present report records the recovery of F.M.D. virus from the air of loose boxes containing infected cattle; it also describes the infection of cattle as a result of exposure to experimentally generated aerosols of the virus MATERIALS

AND

METHODS

Cattle. The animals were fully susceptible Devon or Devon-cross steers. ahout 18 months old, which were purchased through a dealer who collected them from various parts of SW. England. Accommodation. All cattle were housed in vermin-proof loose boxes (12ft. x 12ft. x 9ft.). virus. Cattle-passaged strains of several different immunological types were used to ensure that viability in an aerial suspension was not confined to a single strain. Air sampling. Air in the loose boxes was sampled for virus by being drawn through critical-orifice impingers of a type basically similar to the “Porton impinger” (Henderson, 1952). These were designed and made at Pirbright. Croups of impingers, each containing 10 or 20 ml. of M/25 phosphate buffer (pH 7.6) were operated in parallel to sample metered air volumes of up to 85 cu. ft./hr. Concentration of virus. Virus collected by the impingers was concentrated by pooling the contents and then extracting the virus by adsorption onto finely divided magnesium silcate clay (Attaclay*), which was added to the fluids at a concentration of 1.0 mg./ml. After thorough shaking, the adsorbed virus was deposited by centrifugation and re-suspended in a small volume of supernatant. After the addition of penicillin, streptomycin and mycostatin the suspension was inoculated into groups of mice. Mice. Unweaned albino mice of the Pirbright ‘P’ strain were used for infectivity titrations by the method of Skinner (1951) and for the detection of virus in extracts of impinger fluids. Complement fixation (C.F.) tests. To confirm that deaths in inoculated mice were caused by F.M.D. virus of the type secreted by the cattle, the carcases of paralysed mice and of mice dying later than 24 hours after inoculation were collected, skinned, triturated as aseptically as possible and used as antigen in complement fixation tests (Brooksby, 1952). Part of the carcase suspension was also sub-inoculated into further groups of mice. Serum-virus neutralization tests. Antibody levels in the sera of vaccinated cattle were determined bv the method of Skinner (1953), results being expressed as the neutralization index @.I.) of the serum sample.’ Aerosol generator. The apparatus used to infect cattle by exposure to airborne virus was similar to that described by Hyslop (1963). RESULTS

Detection of Airborne F.M.D. Virus in the Vicinity of Infected Cattle The initial observations on sampling air from loose boxes containing infected cattle indicated that it was necessary to employ the “Attaclay” concentration method. By this technique the virus extracted from about 170 cu. ft. of air was concentrated into a volume of 2.5 ml. and then was distributed parenterally

among 30 to 50 mice. The inoculation of control suspensionsof sterile Attaclay was without l

Attapdgus

apparent Co.,

U.S.A.

effect on similar

groups

of mice.

N.

ST.

G.

121

HYSLOP

In the early experiments, the impingers were operated continuously for relatively long periods at 60 cu. ft./hr., the fluids being collected after alternate sampling periods of 17 hours and 7 hours for about 1 week. The results of an experiment of this type in a loose box containing 2 steers infected by intradermal inoculation of the tongue with type SAT 2 virus are shown in Table 1. It will be seen that virus was detected in the impinger fluids by the 24th hour, at which time primary vesicles were present on the tongues of the cattle. The virus recovered from the air was of the same type as that inoculated into the cattle, and it reached a peak concentration during the 3rd day. 1

TABLE RELATIONSHIP

Cattle

infected

BETWEEN

EARLY

CLINICAL

with Type SAT 2 virus: sampling air for alternate

Sampling period (hours aftr

Clinical

inoculation

SIGNS

OF

F.&D.

Primary tongue

vesicles developing at 24 hr.

24-48

Vesicles

48-72

Secondary

72 - 96

Secondary vesicles on all feet

96-

on

extending vesicles

developing

113 N.A.

AND

MORTALITY

Mouse mortality

condition of cattle

of cattle) O-24

IN CATTLE

mice inoculated with concentrates of impinger periods of 7 and 17 hours at 60 cu. k/hr.

-

Result

IN

MICE

fluids

after

Result of C.F. .test on mouse carcase

Total

%

12198

12.2

N.A.

25/100

25.0

SAT

2

30/100

30.0

SAT

2

14189

15.7

SAT

2

12150

24.0

SAT

2

not available.

In later experiments, in order to reduce virus losses in the impingers, the sampling period was reduced to 2 hours and subsequently to 1 hour. The air flow rate was increased to 85 cu. ft./hr. Virus of several strains of type SAT 1 was used instead of type SAT 2 in most of these air sampling experiments. Table 2 shows that a considerably greater virus recovery rate was achieved. Here again, virus was recovered from the air as early as 18 to 20 hours after inoculation of the steers, and heavy concentrations of virus were present on the 5th day. In a further series of experiments, a small volume (1 ml.) of the pooled impinger fluids was removed for titration in mice before the samples were treated with Attaclay. However, with the strains examined up to the present time, the sampling technique did not collect sufficient virus to permit accurate titration of unconcentrated impinger fluids on the basis of the 50 per cent. infective dose for mice. Results are shown in Table 3. Nevertheless, in experiments employing the concentration method, it has been possible to detect virus in the air of the loose boxes before the vesicles characteristic of F.M.D. appeared on the tongues of the infected cattle. Indeed, airborne virus of strain Israel 4161 was detected during the period immediately after inoculation of virus into the tongues of the animals, i.e. whilst they were still lying anaesthetised on the floor of the box. Unfortunately, in the first experiment in this series it was

122

AIRBORNE

INFECTION

WITH

F.

AND

M.

VIRUS

not possible to confirm the specificity of the earliest mouse deaths by C.F. tests, but the mortality was associated with typical paralytic signs and there is little doubt that death was caused by F.M.D. virus. A fairly high proportion of deaths (g/50) occurred in mice inoculated with virus collected during the period 18 to 20 hours, before vesicles appeared, and the identity of the virus collected at this period was confirmed by C.F. tests. In the second experiment of the series shown in Table 3, virus of a different strain (SA.13/61) was used and the collection period was reduced to 1 hour. Results are very similar to those of the previous experiment; virus was recovered from the air very soon after inoculation of the steers, and on this occasion its TABLE RELATIONSHIP

Cattle

BETWEEN

infected

EARLY

CLINICAL

SIGNS

2 OF F.M.D.

with Type SAT 1 (Strain SA. 13/16) virus: impinger fluids after sampling air for Z-hour

Sampling period (hours after inoculation of cattle)

Clinica~~t~5tion

CATTLE

AND

MORTALITY

IN

.~ou.re mortality

of

of

Result of C.F. test on mouse carcase

Total

7;

20150

40.0

SAT

1

Early

23-25

Vesicles

well developed

26150

.52.0

SAT

1

42-44

Vesicles

extensive

29140

72.5

SAT

1

47 - 49

Feet painful

35140

87.5

S.AT

1

95 - 97

Foot

21130

70.0

SAT

1

21123

91.3

SAT

I

121

vesicles

on tongues

.MICE

mice inoculated with concentrates periods at 85 cu. ft./hr.

18-20

119-

vesicles

IN

ruptured

Healing

TABLE RECOVERY

OF TYPE

SAT 1 VIRUS

85

Strain

inoculatid into cattle Isr.4161

Sampling period (hours after inoculation of cattle) -2

to 0 0 to 2 18 to 20

23 to 25 43 to 45 SA.13161

-2 0 17 24 42

to to to to to

0 1 18 25 43

48 to 49

IN CU.

POOLED FT./HR.

Clinical condition of cattle

Uninoculated Narcotized Blanching of tongue epithelium Vesicles developing Vesicles extensive and rupturing Uninoculated Narcotized No lesions Vesicles present Vesicles extensive and rupturing Feet painful

3

IMPINGER TOTAL AIR

FLUIDS FLOW

AFTER

SAMPLING

AIR

Mouse mortality after inoculation with: ’ Attaclay’ ImpingerJuid diluted 10-0.5 ,@I.0 extract 100.0 -_____ 1/50* 3/50t 9150 12!19 8!20

l/16 2112

AT

Result of C.F. test on mouse carcase N..4. N..4. SAT

1

O/l6 o/12

SAT S4T

t

SAT

1 1 1

o/50 1148 1>40 8130 5/30

o/20 o/13

O/l0 o/10

016 o/10

N.A. N.A. SAT

-

l/20

l/20

o/10

SAT

* Killed

and eaten by dam on 6th day after inoculation. arently specific deaths on 2nd, 4th and 5th days after &?pKesult not available. . .

inoculation.

N.

ST.

G.

123

HYSLOP

identity was confirmed by C.F. tests. Direct titration, though demonstrating the presence of virus, again failed to provide a quantitative result in terms of a 50 per cent. mouse infectivity end-point. Little is known of the efIect of environmental conditions upon the recovery rates of airborne virus. Table 4 shows the results of an experiment made under apparently nearly optimal environmental conditions of cold humid weather with overcast skies. Again, a relatively heavy concentration of virus was present before the disease was obvious to clinical inspection, whilst a 74 per cent. mortality occurred in mice inoculated 4th concentrates of air samples collected as late as the 1 lth day, and mouse mortality remained at 58 per cent. on the 14th day. 4

TABLE DURATION

Virus S4ling

recovered

OF

AIRBO~B

from

P.M.D.

conccntratcs

vmUs

OF TYPE

of pooled impinger overcast weather.

SAT 1 fluids

(STRUN

after

RV. 11/37) sampling in cold,

Mouse

pm’od

(hours ajb

Clinical

condition of cattle

fy%%

mortality TOtill

%

. . on mouse carcase

39140

97

SAT

I*

48150

96

SAT

1

healing

42150

84

SAT

1

165 - 167

Lesions healing

45/50

90

SAT

1

261 - 263

Lesions

healing

37150

74

SAT

1

330 - 332

Lesions

healing

29150

58

SAT

1

inoculation of caw 18-20

N&mo~u

vesicles

96-98

Vesicles

on all feet

114-

116

Lesions

*Preferential

present

fixation

until

with

RV

.l 1 antiserum.

,Znfection of Cattle with Experimentally-generated

Aerosols of F.M.D.

Virus

Groups of cattle were exposed to aerosols generated by a pump-driven reflux nebulizer operating at an airflow rate of about 10 litres/minute. The nebulizer orifice was connected to a face mask by 35 ft. of * in. diameter plastic tubing. The virus suspension, the nebulixer and the generator operator all remained in a separate laboratory and they were not allowed to come into contact with the cattle or with the cattle accommodation. Persons handling the tubing, the mask or the cattle did not enter the laboratory. At the end of the period of exposure, the area of the muzzle covered by the face mask was swabbed well with formalin solution, the animals were removed from the potentially contaminated loose box in which the experiment was made, and were placed singly in clean boxes. To demonstrate that infection was caused by virus in true aerial suspension, and not either in “Fliiggedroplets” or adsorbed onto dust particles, some of the cattle were exposed to an aerosol which had passed through either glass-fibre or 44 cotton-asbestos filter pads inserted between the generator and the face mask. The majority of exposed cattle became infected and virus in samples of tongue epithelium collected from reacting animals was shown by the C.F. test to be of the same type as the virus suspension used in the nebulixer reservoir.

124

AIRBORNE

INFECTION

WITH

OF CAlTLE

Duration Animal

of

T O AEROSOL

OF

WhetherJilter in circuit

e%pOSUre

(minutes)

NO.

DE

EXPOSED

AND

M.

VIRUS

5

TABLE

REACTION

F.

F.M.D.

VIRUS

Clinical

OF TYPE

‘0’

Result of C.F. test

result

of exposure

on tongue

epithelium

69

10

Yes

F.M.D.

on 3rd day

Type

0

80

10

Yes

F.M.D.

on 3rd day

Type

0

on 6th day

Type

0

71

5

No

F.M.D.

73

5

No

Not infected

77

5

NO

F.M.D.

on 2nd day

Type

0

78

5

No

F.M.D.

on 4th day

Type

0

6

TABLE REACTION

Animal NO.

OF

VACCINATED

AND UNVACCINATED VIRUS OF TYPE ‘C’

Zmmune status at time of exposure

-

CATTLE EXPOSED FOR ti MINUTES

Whether filter in circuit

TO

Clinical

AEROSOL

result

of exposure

OF F.M.D.

Result of C.F. test on tongue

epithelium DB

89

Vaccinated: N.I.* = 2.145

Yes

Not

infected

DB

90

Vaccinated: N.I.* = 2.670

rio

Not infected

DC

43

Susceptible

Yes

F.M.D.

on 7th day

Type

C

DC

44

Susceptible

No

F.M.D.

on 4th day

Type

C

* N.I.

= Virus

neutralisation

index

of serum.

Table 5 shows the results of a typical aerosol exposure experiment. It will be seen that 5 of the 6 cattle exposed became infected with virus of type “0”. As the inclusion of filters of the type used to retain dust and the larger bacteria did not affect the results, it may be concluded that infection was caused by a true aerosol either of very small droplets or of droplet nuclei. Table 6 shows a similar experiment with virus of type “C”. In this experiment, 2 of the cattle had been vaccinated and the neutralization indices of their sera, determined by the virus neutralization test described by Skinner (1951), were sufficiently high to have protected the animals against parenteral challenge. Both vaccinated cattle were protected against airborne infection whereas both unvaccinated cattle developed F.M.D. DISCUSSION

Hyslop (1965) has demonstrated that large amounts of virus are secreted in the saliva of infected cattle and has also confirmed the observations of early workers that the saliva often becomes infected before clinical signs of F.M.D. appear. Attention was drawn to the possible dangers of aerial transmission.

N.

ST.

G.

HYSLOP

125

The smacking movements of the tongue and the foamy nature of the saliva of cattle early in the acute phase of the disease suggest that dkemination of large numbers of infected droplets may occur almost as soon as the mucosa of the tongue begins to liberate virus. The results of the present investigation lend support to this suggestion. Indeed, small amounts of airborne virus were detected in air samples collected during the first 2 hours after the cattle were inoculated, though there is evidence that any virus which seeps from the needle tracks of the original inoculation is no longer detectable in the oral secretions after 2 to 4 hours. In all the experiments, virus was recovered from concentrates of the impinger fluids used for air sampling at about the 18th hour, and this correlates well with the regular detection of virus in saliva samples collected at the 16th to 18th hour after infection (Hyslop, 1965). The amount of virus recovered from the air is smaller than would be expected, having regard to the high concentrations which are detectable in the oral secretions of cattle when vesicular lesions are present. This may be due to the aerial dilution or to inefficient recovery. Thus, sampling for long periods may cause loss of viable virus from the impinger fluids as a result of inactivation by heat or by re-nebulization consequent upon prolonged bubbling in the impingers; the various causes of viability loss in bubbler systems were reviewed by Rosebury (1947). Furthermore, gradual solution of ammonia present in the atmosphere of the loose box eventually overcomes the action of the buffer and causes a rise in pH of the fluids, which may be sufficient to exert an adverse effect on the virus. Variation in the sensitivity of unweaned mice, used as indicators, for different strains of virus (Heatley, Skinner and SubakSharpe, 1960 ; Subak-Sharpe, 1961) also may have considerable influence on the recovery rate for a particular strain as compared with others. Changes in the environmental conditions, such as fluctuations of temperature, humidity, sunlight and minor air currents, may be at least as important as sampling factors. In the last experiment the mortality rate in groups of mice inoculated with concentrates of the impinger fluids remained in the range 84 to 97 per cent. from the 20th to the 167th hour. As in the previous experiments, virus was detected before lesions were evident and a fairly high mortality rate was observed in mice inoculated with a concentrate of the sample collected about 14 days after the cattle were inoculated. It is believed generally that the saliva of cattle does not remain infective for periods longer than 4 to 5 days after the develop ment of lesions and, since all the lesions were healing well by the 12th day, it is apparent that much of the virus recovered from the air may not have been secreted recently. It is probable that the greater part of the virus collected in the latest samples had been swept into the air from the walls, the floor and from the coats of the animals. In spite of reservations regarding the immediate source of virus, however, the effect of the observations remains unchanged : cattle infected with F.M.D. may contaminate the air in their immediate vicinity with virus for periods of at least 2 weeks, The previous experimental evidence for “airborne infection” suggested that virus might be disseminated most readily by adsorption onto dust or hay particles etc., instead of in true aerial suspension of droplets and, since all but the smallest dust particles would fall to the ground fairly rapidly, such spread might be confined principally to the immediate vicinity of infected cattle. In the present experiB

126

AIRBORNE

INFECTION

WITH

F.

AND

M.

VIRUS

ments, the successful infection of cattle as a result of the inhalation of infected air, which had been passed previously through filters capable of retaining dust, indicates that airborne micro-droplets, or their nuclei, are also a potential source of infection. Further quantitative studies are necessary to assess precisely the relative importance of airborne infection and to determine the longevity of virus particles in the airborne state. CONCLUSIONS

Although the virus of foot-and-mouth disease (F.M.D.) spreads most frequently by either direct or indirect contact with infected animals, an accumulation of evidence from field sources suggests that cattle may be infected occasionally by airborne virus. Air in loose boxes containing experimentally infected cattle was sampled by “Porton-type” impingers; virus collected from the air was concentrated by adsorption and was detected by intraperitoneal inoculation into unweaned mice. The method was successful regularly in detecting airborne virus before clinical signs of F.M.D. were evident; thereafter virus could be recovered from the air for periods of up to 14 days. Several factors, including environmental conditions, appeared to influence the rate of recovery of virus. Experimentally generated aerosols of F.M.D. virus infected susceptible cattle and it was concluded that airborne spread of virus may occur under natural conditions in the field. ACKNOWLEDGMENT

The author wishes to thank Mr. R. L. G. King for his valuable technical assistance. REFERENCES

Brooksby,

J. B. (1952). The Technique of Complement-Fixation in Foot-and-Mouth Disease Research, A.R.C. Rept. Ser. 12, H.M. Sta. Off.; London. Fogedby, E. G., Malmquist, W. A., Osteen, 0. L., and Johnson, M. L. (1960). Nord.

Vet Med.,

12,490.

Heatley, Wendy, Skinner, H. H., and Subak-Sharpe, H. (1960).Nature,

909.

Lond.,

186,

Henderson, D. W. (1952). J. Hyg., Camb., 50, 53. Hyslop, N. St. G., and Ford, J. (1957). Vet. Rec., 69, 521. Hyslop, N. St. G. (1963). J. camp. Path., 73, 265; (1965). Ibid., 75, 111. Jerlov, S. (1940). Vet. Bull. (Weybridge), 11, 511. McLean, R. C. (1938). Nature, Lond., 141, 828. Moosbrugger, G. A. (1948). Schw. Arch. TierheiEk., 90, 176; (abs. Vet. Bull., 19, 479). Rosebury, T. (1947). Experimental Airborne Infection, Williams and Wilkins; Baltimore. Skinner, H. H. (1951). PYOC.soy. Sot. Med., 44, 1041; (1953). Proc. XVth Int. vet. Congr. Stockholm, p. 195. Subak-Sharpe, H. (1960). Arch. ges. Virusforsch., 11, 39. Traub, E., and Wittman, G. (1957). Berl. Miinch. tier&-d. Woch., 10, 205. [Received

for

publication,

September

3rd, 19641