1959 viruses

J.COMP.PATH.

1967. VOL. 77.

EXPERIMENTAL INFLUENZA

159

INFECTION

OF

A/TERN/SOUTH

CHICKENS

AFRICA/1961

CHICKEN/SCOTLAND/l959 I. CLINICAL

WITH AND

VIRUSES

PICTURE

AND

VIROLOGY

BY

W. B. BECKER Department of Bacteriology and C.S.I.R. and U.C. 1. Virus Research Unit, University of Cape Town Medical School, Republic of South Africa and c. Department

of Pathology,

Uniuersity

J.

UYS

of Cape Town Medical

School, Republic of South Africa

INTRODUCTION

Influenza A/Tern/South Africa/l961 virus (TV) was shown to be the causative agent of an epizootic with a high mortality rate in the migrant European Common Tern (Sterna hirundo) in South Africa in 1961 (Becker, 1962). Influenza A/Chicken/Scotland/l959 virus (CV) caused a fowl-plague-like outbreak in chickens in Scotland in 1959 (J. E. W’lI son, personal communication, 1962). The isolation and classification of TV as an avian strain of influenza A, which had a strain-specific antigen distinct from other known influenza A viruses with the single exception of CV, have been described and the interesting epidemiological possibilities raised by this close antigenic relationship have also been discussed (Becker, 1966). D u&g the epizootic the danger existed that TV might spread from the terns to the many poultry flocks in the immediate vicinity. Chickens were therefore infected experimentally with TV to study the pathogenesis of the diseaseproduced, and, in addition, this diseasewas compared with that caused in chickens infected with CV. The purpose of this paper is to present the clinical and virological results. The pathological findings will be reported in a separate communication (Uys and Becker, 1967). MATERIALS

AND METHODS

The autopsy procedure, the preparation of tissuesfor virus studies, the titration of virus in embryonated eggs and the assay of serum neutralizing and haemagglutination-inhibiting (HI) antibodies have been described (Becker, 1966). Virus Strains

InfEuenza A4/Tern/South Africa/l961. TV was propagated in embryonated eggs by inoculatio; n and used at the 8th and 9th passagelevels. Influenza 1 1/Chicken/Scotland/Z959. CV was made avaiIable by Dr. J. E. Wilson* and was employed at the 9th allantoic passagelevel in our laboratory. *Veterinary

Laboratory,

Ministry

of Agriculture,

Lasswade,

Great Britain.

160

INFLUENZA

INFECTION

IN

CHICKENS

:

VIROLOGY

Chickens and Embryonated Eggs Leghorn-Australorpe cross chickens and eggs from a commercial source were employed in the experiments. Except in Experiment I with TV, the chickens used were 6 to 7 weeks old. Their environmental temperature was maintained at 20 to 22OC. RESULTS

Experimental

Tern Virus Infection

(TVI)

in Chickens

Susceptibility of chickens to different routes of inoculation (Experiment I). Groups of 3-week old chickens were inoculated with 0.1 ml. of allantoic fluid containing 10”‘” egg infective doses (EID) 50 of TV intramuscularly (IM), into the conjunctival sac (ICS), intranasally (IN) or orally (PO). Eight controls were inoculated IM with 0.1 ml. of norma allantoic fluid. The groups were caged at least 3 ft. from one another. TV1 was produced b’y each of the 4 routes of inoculation employed. Eight of the 10 chickens inoculated IM died of TV1 within 5 days, another died on day 7 and the remaining chicken on day 10. Six of the 8 chickens inoculated ICS died within 6 days and one on day 13. One was still unaffected on day 17 when it was challenged with 106’6 EID,, of TV inoculated IM; it died of TV1 4 days later. Five of 8 chickens inoculated died within 5 days, a 6th bird recovered after showing mild signs of TV1 from day 9 to 13. Two remained well but died within 5 days of being challenged on day 17 with 1O6’6 EIDso of TV inoculated IM, while the convalescent bird was unaffected by the same challenge inoculum and had a serum HI antibody titre of 20 two weeks later. Four of the 5 chickens inoculated PO died from 23 to 25 days later, but the remaining bird was still unaffected on day 37 and had no HI antibodies in its serum. The controls remained well throughout the experiment. Distribution of virus in the tissues of infected chickens (Experiment 2). Twentyfour chickens were inoculated with 106+ EIDao of freshly harvested TV. Half the inoculum of 0-l ml. was introduced into the right conjunctival sac and half into the right nostril. The chickens were placed in 24” x 18” x 12” cages, six to a cage. Six control chickens were inoculated with normal allantoic fluid and caged 3 ft. from the other chickens. Starting the day after infection, a batch of chickens was autopsied each day for six days. The two convalescent survivors were examined on day 10. One control chicken was autopsied with each batch except on day 4. Suspensions (10 per cent. w/v) of pectoral muscle, liver, spleen, lung, kidneys, heart, brain and blood were prepared for virus studies and stored at -2OOC. The virus contents of a given tissue of all the chickens were titrated concurrently or in two batches within four days of each other. All the titrations were completed within four months of the start of the experiment. Portions of the above organs, and of comb, soft palate, skin, small intestine, renal adnexae and both eyeballs with their eyelids were taken for histological examination. In chickens autopsied one day after inoculation virus was only detected in the kidney. A transient viraemia associated with widespread distribution of virus in the tissues occurred early in the course of the disease. Thereafter virus was detected almost invariably in the brain, heart and lung and not infrequently in breast muscle. Serum HI antibodies were demonstrated in one of the chickens harvested on day 5, but virus was still present in its tissues (Chicken 20, Table 1).

W.

B.

BECKER

AND

C.

J.

UYS

161

162

INFLUENZA

INFECTION

IN

CHICKENS

:

VIROLOGY

Both ,convalescent chickens autopsied on day 10 had antibodies and their tissues had apparently been cleared of virus (Chickens 23 and 24, Table 1). All the infected chickens showed histological evidence of TV1 except for those autopsied on day 1 and Chicken 9 (Table 1) which appeared to escape infection completely because no virus or lesions were detected in its tissues nor antibodies in the serum. The controls yielded no evidence of infection with TV. Signs and course of TVI. Twenty-eight chickens were inoculated with TV as in Exp. 2 and the signs and course of the disease were observed. Six moribund chickens were autopsied on the 5th day after inoculation and another on day 12. Two of three surviving chickens were autopsied on the 19th day and the 3rd on day 25. Two control chickens inoculated with normal allantoic fluid were autopsied, one on the 4th and one of the 25th day. Suspensions of blood, heart, spleen, lungs and brain were stored in a dry ice cabinet and the virus contents titrated within a month of their preparation. A portion of each of the above organs and the proventriculus and ventriculus were taken for histological examination (Exp. 3). The incubation period was 3 to 5 days and the chickens usually died within 48 hours of the onset of illness. Most of the 28 chickens died within a week of inoculation, but 3 died on the 11 th or 12th day, two recovered from the illness and one chicken showed no evidence of infection. The earliest signs of TV1 were listlessness, ruffling of the feathers and retraction of the head, but the affected chickens appeared normal when roused. Some chickens had ventral soiling due to loose stools. Within several hours the affected chickens developed a “dozing-off” appearance with drooping head and wings, and sometimes preferred to squat. At this time the palpebral conjunctivae were congested and on the conjunctival surfaces of the lower eyelids towards the inner canthus pin-head sized, white nodules stood out prominently. Some chickens continued to eat and drink though ill. One -or more of the following signs developed :-paresis, tremors, muscle spasms and convulsions. The chickens became lethargic and usually lay in bizarre spastic attitudes, sometimes exhibiting slow spastic or convulsive movements. Some chickens remained in this state for about 24 hours before death. The combs were congested and cyanosed. In chickens surviving for 5 days or more, ulcerating lesions developed on the combs and sometimes on the eyelids, and healed slowly in birds which recovered. There were no signs or respiratory difficulty in any of the chickens, nor was there any discharge of secretions from the nasopharynx. A few chickens showed mild signs of late onset followed by recovery and the occasional chicken appeared to be refractory to infection. The controls remained healthy throughout. Virological findings. Higher concentrations of virus were demonstrable in this experiment than in Exp. 2 because of the more favourable storage conditions of the tissues. Virus was detected in all 6 moribund chickens autopsied on the 5th day. The titre was high in the brain, less so in the heart and lungs, but no virus was detected in the blood or spleen. There was histological evidence of florid TVI. Virus was detected in the palatal swabs and/or cloaca1 swabs of 5 chickens which were tested. The serum of only 1 of the 6 chickens (Chicken 5, Table 2) contained HI antibodies. The moribund chicken harvested on day 12 (Chicken 7, Table 2) had apparently overcome the active phase of TV1 as no virus was demonstrable in

W.

B.

BECKER

AND

C. J.

163

WS

samples of its tissues taken at autopsy and the histological lesions of TV1 showed signs of regression. No virus was isolated from two convalescent chickens (Chickens 8 and 10, Table 2) autopsied on the 19th and 25th day after infection and whose sera contained HI antibodies. The former showed residual minimal histological lesions but no lesions were detected in the latter. Chicken 9 (Table 2) apparently escaped infection as it remained well, no virus or lesions were detected in the tissues, nor antibodies in the serum. The control chickens showed no evidence of TVI. Experimental

Chicken

Virus

Infection

(CVI)

in Chickens

(Experiment

4)

The purpose of the experiment was to note the signs and course of CVI and to determine the distribution of virus in the tissues of sick chickens. Thirty chickens were inoculated with 1O”‘5 EIDsO of freshly harvested CV administered TABLE EXPERIMENTAL

Day

TERN

content

2

INFECTION

after inoculation

IN

CHICKENS,

3

EXPERIMENT

5

Chicken no. Virus

VIRUS

1.2

19

7

25

1

2

3

4

5

6

8

9

10

6-o* 2”:;

7-7 5.7 4.3

6-3 4.0 4.2

7-5 *.g 4.7

7-7 5.5 5.8

r -

I -

17 -

-

-

7-5 5.7 3.5 -

K -

_I -

+ -

7

1

NT

40

7

ii

ofBlood Brain Heart Lung Spleen

Palatal swab Cloaca1 swab Serum HI antibody

titre

;IT;I--+ -

*log.,,ID,, per g. - = no virus detected, += virus present. NT = not tested.

or no antibodies

detected.

by the same routes used in Exp. 2, and distributed in 24” x 18” x 12” cages, 6 to a cage. Three sick chickens were autopsied on day 3, a further three on day 4, and a convalescent chicken on day 14. One uninoculated running-in control was placed in each cage of chickens inoculated with CV and three control uninoculated chickens were placed in a separate cage in the same room. Six chickens were inoculated with lo”.” EIDu, of TV and caged on their own. Suspensions of blood, heart, lungs and brain were prepared from each autopsied chicken, stored in a dry ice cabinet and titrated for virus content within four weeks of preparation. Portions of heart, lungs, brain, liver, spleen, kidney and adnexae, skin, comb, proventriculus ventriculus and small gut were collected for histological examination. Course of the illness. The incubation period was 2 to 5 days. The first sign was listlessnessand death usually followed rapidly. Twenty-one of the 30 inoculated chickens died on the 3rd or 4th day after inoculation; most of them appeared well in the evening but were found dead the next morning. Four chickens survived for 2 to 4 days after the first sign of illness and died 5 to 8 days D

164

INFLUENZA

INFECTION

IN

CHICKENS

: VIROLOGY

after inoculation. Two chickens recovered and developed serum HI antibody titres of 80 and 160. Three apparently escaped infection as they remained well and did not develop HI antibodies. In contrast with TVI, no tremors, spasms or convulsions were noted. A distinctive feature, not seen in TVI, was congestion of and haemorrhage into the superficial tissues. This was most evident in the skin of the legs around the joints, on the conjunctival surface of the lower lid and in the cloacal mucous membrane. The combs were congested and cyanosed and ulcerating lesions developed on the combs and sometimes on the eyelids if the illness ran a subacute course. Virological investigations. The virus content of the blood and tissues of the sick chickens was moderately high, there were no HI antibodies in the sera and all the birds showed histological evidence of infection (Chickens 1-6, Table 3). No virus was detected in the convalescent chicken (Chicken 7, Table 3), which showed healing lesions histologically and a serum HI antibody titre of 160. TABLE EXPERIMENTAL

CHICKEN/SCOTLAND

VIRUS

3 INFECTION

Inoculated Day Chicken

Virus

of experiment

content Blood Brain Heart Lung Serum HI antibody titre

I

EXPERIMENT

4 3

2

4 Sick contact control

chickens

3

no.

IN CHICKENS,

4

5

3.25 1.75 1.75 4.0

3.25 4.0 4.5 7.5

3.0 3.0 3.0 4.5

-

_

_

14

9

6

7

8

4.0 3.25 3.25 >4.5

--

>3.5 3.5 3.75 >4.5

-

ofNT 4.25 6.0 7.25

;:“o* 4.25 >4.5

NT

-

NT

* log.,,ID,, per g. = no virus detected, = not tested.

or no antibodies

160

NT

detected.

Control uninoculated chickens. One contact control (Chicken 8, Table 3), first showed signs of CVI on day 7 and died within 36 hours. All the 6 inoculated cage companions had developed CVI but 2 recovered. One of the 3 uninoculated chickens caged on their own was autopsied on day 3 and showed no evidence of CVI. All the other uninoculated chickens remained well and had not developed antibodies when the experiment was terminated on day 25. Control Chickens Inoculated with TV Five of the 6 chickens developed TV1 and showed the same clinical and virological features noted in Experiments 2 and 3. The 6th chicken apparently escaped infection. DISCUSSION

Experimental infection of chickens with Tern virus produced an acute clinical disease with a high mortality rate similar to the disease seen in Common Terns in the epizootic of 1961. The experiments were not designed to imitate field con-

W.

B.

BECKER

AND

C.

J.

UYS

165

ditions, but chickens were successfully infected with TV inoculated intranasally, into the conjunctival sac, orally or intramuscularly, thus indicating possible routes of infection. Possible sources of infection from sick chickens were the nasopharynx, cloaca1 secretions or excretions and exposed tissues. Chickens infected experimentally with chicken virus developed a similar clinical picture to that noted in the natural outbreak in chickens in Scotland in 1959 (J. E. Wilson, personal communication, 1962). The present study showed that experimental TV1 and CVI could be distinguished from each other despite the close antigenic relationship between the two virus strains shown previously (Becker, 1966). The duration of the clinical illness in TV1 was longer and there were some signs such as paresis, tremors, muscle spasms and convulsions, which were not seen in CVI. Furthermore, congestion of and haemorrhage into the superficial tissues were distinctive signs of CVI. At the time of death of chickens from TVI, virus was most consistently demonstrable in the brain, heart and lungs and viraemia was not a feature. In CVI, viraemia was a constant finding in chickens at the time of death. It may be noted that TV was isolated from a few chickens even after the appearance of antibodies in the serum. This observation has also been made in Newcastle disease by Sinha, Hanson and Brandly (1952). SUMMARY

Chickens were readily infected with Tern virus/South Africa/l961 inoculated by the conjunctival sac and intranasal routes. Three to five days after inoculation most of the chickens developed an acute illness and died within 48 hours. Virus studies showed an early transient viraemia after which virus was most consistently demonstrated in the brain, heart and lungs. The disease could be distinguished from that produced by Chicken virus/Scotland/l959 despite the close antigenic relationship between the two viruses. ACKNOWLEDGMENTS

We are indebted to Professor A. Kipps for his advice, and would Misses E. Baker and S. van Olm for able technical assistance.

like to thank

REFERENCES

Becker, W. B. (1962). S. Afr. J. lab. clin. Med., 8, 163; (1966). J. Hyg., Camb., 64, 309. Sinha, S. K., Hanson, R. P., and Brandly, C. A. (1952). J. infect. Dis., 91, 276. Uys, C. J., and Becker,

W. B. (1967). J. camp. Path, 77, 167. [Received

for publication,

July 9th, 19661