Research in Veterinary Science 1991, 50, 222-228

Effects of recombinant human o~A interferon in gnotobiotic calves challenged with respiratory syncytiai virus M. J. DENNIS, L. H. THOMAS, E. J. STOTT, AFRC Institute for Animal Health, Compton,

Newbury, Berkshire

The effects of recombinant human c~A interferon were studied in gnotobiotic calves challenged with respiratory syncytial virus (Rsv). Gnotobiotic calves given doses of interferon by intramuscular injection over five days showed a marked, dose-related, rise in rectal temperature and depression of circulating leucocytes. Differential counts showed decreases in both lymphocytes and neutrophiis. No significant pathological differences were found between treated and untreated calves, nor could any difference be demonstrated in the pattern of RSV infection.

Escherichia coli, containing less than the permissible level of E coli protein (20 ng mg- i 1EN) agreed with

the National Institute for Biological Standards and Control (Hoffman la Roche), was administered by intramuscular injection in the hindquarters over a period of five days (day 0 to day 4). In the first experiment, calf V70 was given an initial dose of 36x 106, or 36 megaunits (MU), of 1FN (0"45 MU kg -1 bodyweight) at 09.00 on day 0. This produced a pronounced clinical reaction and no further interferon was administered on that day. A reduced total daily dose was subsequently adminisTHE effects of human leucocyte interferon (HulFNc0 tered, that is, 18 MU three times per day spread over preparations have been noted in human volunteers a period of 12 hours (09.00, 15.00 and 21.00). This (Scott et al 1981, Scott 1982). Bovine cells are highly dose regime was followed for a further four days (day sensitive to HulFNc~,thus the positive effect of HulFNc~ 1 to day 4). In the second experiment a dose regime of three has been tested in calves against vaccinia virus (Werenne et al 1983) and against infectious bovine injections of 18 MU (0.225 MU kg-l) of interferon rhinotracheitis virus (Roney et al 1985), while Cocker per day was followed from day 0 to day 4 on calf et al (1987) studied the effect of the HulFN,~ on the V126. course of feline viral rhinotracheitis. The availability of recombinant bovine IFNc~ has led to studies by Samples Gillespie et al (1986a,b) and Bielefeldt Ohmann and Blood samples were taken three days before the Babiuk (1986) on its effect in calves. The purposes of this experiment were, first, to start of IFN administration and from day 0 to day 7 study in detail the clinical effects of HuIFN~xon gnoto- for haematology, serum IFN levels, serology and biotic calves and, second, to look at the effects of plasma zinc and copper estimations. Rectal temHulFNc~Aon the course of an experimental infection peratures were measured at regular intervals over the same period. with respiratory syncytial virus (RSV) in calves. Nasopharyngeal swabs were taken before challenge with RSV and daily to day 7. Materials and methods Tracheobronchial lung washes were made with Animals 150 ml phosphate buffered saline on day 3 and day 7, Four Friesian calves, V70, V72, Vl15 and V126, on the conscious animal, using an endobronchial were derived as described by Dennis and Wheelock catheter (5 mm diameter) passed through the nasal (1978) and reared under gnotobiotic conditions in cavity into the trachea with the aid of a previously inserted larger guide tube (10 mm diameter). plastic isolators (Dennis et al 1976). Experiments were conducted on pairs of threemonth-old calves, weighing approximately 80 kg, on two separate occasions with one calf in each pair (V70 and V126) receiving interferon.

Interferon HulFNc~Aderived by recombinant DNA synthesis in

Assays Red blood cells were diluted in formol-citrate solution and white blood cells in 2 per cent acetic acid. Cell counts were estimated by using a Neubauer haemocytometer chamber. Packed cell volumes (PCV) were estimated by using a microhaematocrit centrifuge.

222

223

HuIFNc~ in RSV challenged calves Differential leucocyte counts were made on smears stained by Leishmann's solution. Copper and zinc levels were estimated from plasma samples by atomic absorption (Taylor and Bryant 1981). w~ in serum and lung washings was assayed by Dr T. Krummenacher at Hoffmann la Roche, Basle, by antiviral activity and by interferon c~immunoradiometric assay in which WN~ specifically binds to sheep immunoglobulin immobilised on polystyrene beads (Celltech). Direct quantitation of the IFN bound to the solid phase was achieved by the addition of ~2~I-labelled monoclonal antibody NK2 which binds to a second epitope on the IFN molecule. Gamma counts for samples were compared to a standard curve. Methods of virus isolation were as described by Stott et al (1984).

10,000-

Inoculum The Snook strain of ~sv (Thomas et al 1984) was grown in calf kidney cells to produce a stock with a titre of 1 0 4 pfu m l - ~. Ten ml of this inoculum were given both intranasally and intratracheally to all four calves at 14.00 on day 0. Pathology Calves were killed by intravenous injection of sodium pentobarbitone on day 7. The lungs were taken for histological and virological examinations. Results

A distinct febrile response was seen in both calves

10,000~)

b

a

E

---~

oo

,,, & ~\

~' ,,, ¢"

,,,

o I-2000

!

!

=

|

i

|

!

g

i

i

2000

200 -

200 -

20,

20.

i E

z

,, ,, z~ Z

',,

#/-~'--.z~._-- ~

&.,,

2 !

i

i {~

i

!

u

i

i

!

i

41-

,o o

40-

4O

39-

39-

38-

38-

'~__--~.. i 4 i ",x~, ~.4., ;' -...} ' , # s s.~ ~

"~". . . .

6 37,

_3 u 2 u

tit n ttt1 1ttt ttt wttt 2 n3 4 u5

1 nO

u 6

u 7

37

_31 .2=

_ln

0 ~ 1

Days FIG 1 : L e u c o c y t e c o u n t s and rectal t e m p e r a t u r e s in relation t o serum IFN. (a) IFN t r e a t e d c a l v e s • " IFN to b o t h c a l v e s , I IFN to V 1 2 6 only. (b) /~ -- -- - - A u n t r e a t e d c a l v e s - - mean v a l u e s

I 2 w 3 ~ 4

u 5 ~ 6 u 7u

Days

• V70, O--

-- - - © V 1 2 6 .

224

M. J. Dennis, L. H. Thomas, E. J. Stott

following treatment with HuIFNc~(Fig I a). In calf V70, As in V70, the highest level of IFN (147 iu ml l) which received twice the initial dose compared with was recorded on day 2 but the pattern of daily peaks V126, the response was more pronounced with a was not as distinctive and IFN administration on day 4 recorded peak rectal temperature of 4 0 . 5 ° C four produced no increase in serum IFN levels. In general hours after treatment. With no further IFN given the • EFN levels were maintained at a higher level than in V70. temperature began to decline but was still above normal (39.9°C) at 21.00, that is, 12 hours after Interferon was first detected in the serum of control treatment. At 09.00 on the following day, the temcalves at 21.00 on day 1, that is, 36 hours after perature had fallen to 38" 8°C. challenge with Rsv (Fig l b). Subsequent serum A similar initial response was seen in V126 which samples showed endogenous IFN to be present at developed a rectal temperature of 40"2°C four hours varying levels for the rest of the experiment but with after the first dose of IVy. With subsequent doses of levels fivefold to 10-fold lower than those of 1FN IFN on day 0 the temperature continued to rise and a treated calves. recorded maximum of 40.7 oC was observed at 21.00. Interferon was detected in lung washes taken on As in V70 the temperature at 09.00 on the following days 3 and 7 from all calves (not shown). Levels varied morning had fallen to 38.6°C (12 hours after the last but mean levels of IFN treated calves were 32 iu m l - i dose of IFN). on day 3 and 15 iu m l - l on day 7. In untreated calves Subsequent doses of IFN (18 M U for both calves) the mean values were 19 iu ml i on day 3 and 44 iu induced a febrile response which was progressively m l - 1 on day 7. less marked on each successive occasion. Two to three hours after the initial dose of IFN, calf Haematology V70 showed muscular tremors, especially of the hindquarters. The animal also appeared depressed and After administration of IFN, a sharp fall in total apathetic. Six hours after the single dose the animal leucocyte count was seen in both V70 and V126 fed well and by 21.00 (12 hours after treatment) was (Fig la), reaching a minimum at 17.00 on day 0 again bright and alert. Subsequent doses induced (2.15 × 109 cells litre -1 for V70, 2.65 × 109 litre -1 for further apathy and depression which became proV 126). Further falls were seen on subsequent days for gressively less marked over the treatment period. V70 especially on day 3 ( 2 . 8 × 109 litre -1) and V126 Calf V126 showed a similar pattern of apathy and showed a dip late on day 3 followed by another on day 6 (4"0× 109 litre -1 on day 3 and day 6). depression but no muscular tremors were observed. Diurnal fluctuations in rectal temperature were The untreated calves V72 and Vl15 showed no observed in the control calves V72 and V115 (Fig lb) marked leucopenia but there was a drop in mean and need to be taken into account when considering leucocyte count to 4 . 9 8 × 1 0 9 litre -1 on day 3 the febrile response to 1FN. Temperatures, however, (Fig Ib). never rose above 39°C until day 4 when a mean peak Both IFN treated calves showed a drop in lymphoof 3 9 . 2 ° C was observed. cyte levels reaching a minimum count at 17.00 on day 0 giving a mean value of 1 . 4 7 × 109 cells litre -1. Numbers had returned to pretreatment values by the

Interferon assays

None of the calves showed detectable levels of ¿FN in serum before 1FN administration and, or, challenge with Rsv. In calf V70 a sharp increase in serum IFN was seen following the single dose administered on day 0, becoming maximal two hours after administration (Fig l a). Levels declined rapidly after this single injection but were still detectable after 24 hours. Subsequent injections of IFN three times daily on days 1,2, 3 and 4 caused daily peaks of serum IFN with the highest level (221 iu m1-1) recorded on day 2: IFN levels on these days never fell to the 24 hour level following the single dose on day 0. When administration ceased 1FN levels dropped as on day 0 and reached a similar level. In calf V126 a similar response was seen to the initial dose of IFN on day 0, but with further doses on day 0 the serum levels continued to increase.

10,000

-

7

-o9,

~.~"o..

-3'-2'-1'

0

~;~---~

I

90,

--%. o2 Days

FIG 2: D i f f e r e n t i a l l e u c o c y t e c o u n t s . * m e a n v a l u e s IFN t r e a t e d c a l v e s . + mean values untreated calves. A-v a l u e s IFN t r e a t e d c a l v e s . O - - - - - © values untreated calves

.o--°,..

3'4

"~..~___o 5'6

7

* Lymphocytes --- --+ Lymphocytes -A Neutrophils -- mean Neutrophils -- mean

HulFNe~ in RS V challenged calves

p c v values remained depressed until day 4 when an upward trend was observed. Erythrocyte counts (not shown) reflected changes in ecv values. PCV values in control calves showed a steady downward trend reaching minimum values on day 4.

50-

I

q

o

°---41 .......

40

,p,o~

., ;°"cL ,

20

,"~

~

/ /*,, # ,

.

-3

.

-2

.

-1

.

0

225

.

.

I

.

2 Days

Serum, copper and zinc levels

,

/ \ + /

No changes in serum copper or zinc levels attributable to interferon treatment were observed.

,

3

4

5

•

6

7

FIG 3: Packed cell v o l u m e s . - x - - - - x M e a n v a l u e s IFN t r e a t e d calves. '~. . . . . ~ Mean values untreated calves

following day and from then on the mean numbers of lymphocytes followed those of control calves (Fig 2) with a slight dip on day 3 (3" 16x 109 litre-1). Mean lymphocyte values for the control calves showed no great variation until day 3 when a decrease to 3 "61 × 109 cells litre-~ was observed. Neutrophil counts for IFN treated calves began to decline after treatment, reaching minimum values at 21.00 on day 0 and 09.00 on day 1. Cell numbers remained depressed until 14.00 on day 2 when a peak was observed. This peak was followed by a further depression on day 3 and day 4 after which a gradual increase in neutrophils was observed. Neutrophil counts in control calves showed an increase at 13.00 onwards'on day 0, declining to previous values by day 1. A second peak was seen on day 2 followed by depressed numbers on day 3. pcv for IFN treated calves showed a decrease after IFN treatment (Fig 3) falling 6.5 per cent by 17.00.

Pathology

In the two calves treated with IFN, pneumonic consolidation affected under 1 per cent of the lung in V70 and 3 per cent in V126. The extent of consolidation in the two untreated calves, V72 and Vl15, was 5 per cent and nil. No significant difference could be demonstrated, therefore, in the extent of macroscopic lesions between treated and untreated animals. Histological changes in the lungs of all four calves were minimal, comprising essentially an interstitial pneumonitis with infiltration of alveolar walls by round mononuclear cells and patchy atelectasis. Only in very limited areas of the lung were any changes specific for RSV infection recognised. These comprised hypertrophy of the alveolar and small bronchiolar epithelium, sloughing of cells into the lumina and formation of the occasional multinucleate syncytium. Intracytoplasmic phloxinophilic inclusion bodies were also occasionally seen. The bronchial lymph nodes of calf V72 appeared hyperactive in comparison with the nodes of the other three animals. The germinative follicles were prominent, each contained several mitotic figures and the sinuses and paracortical sinuses were filled with large numbers of lymphocytes and macrophages.

T A B L E 1 : Virological findings from gnotobiotic calves after t r e a t m e n t with HulFNe Virus isolation (pfu m l - 1 iog.lO ) IFN Control V70 V126 V72 V115

Immunofluorescence IFN V70

V126

Control V72 Vl15

Nasopharynx Day O 1 2 3 4 5 7

0"7 3"3 3'2 4"2 3'4

0"7 2'6 2'8 3"3 2"0

-1 "6 2"2 3'8 4"0

Day 3 7