Avian Pathology ( 2000) 29, 545–556

Avian pneumovirus infection of laying hens: experimental studies J. K. A. Cook1*, J. Chesher1, F. Orthel2, M. A. Woods1, S. J. Orbell1, W. Baxendale1 & M. B. Huggins1 1

Intervet, UK, The Elms, Thicket Road, Houghton, Huntingdon, Cambs. PE17 2BQ, UK, and 2Intervet International, PO Box 31, 5830 AA, Boxmeer, The Netherlands

Administration of a virulent strain of avian pneumovirus ( APV) to specific pathogen free laying hens by the oculonasal route failed to induce a drop in egg production or any adverse effects on eggshell quality. However, intravenous ( i.v.) inoculation of the same strain caused a substantial drop in egg production and a high incidence of soft and thin-shelled eggs. Some respiratory signs were also observed and the hens appeared sick, with diarrhoea being observed in approximately one-half of the hens between 4 and 11 days post-inoculation ( p.i.). APV antigen was detected in the oviduct epithelium up to 9 days p.i. This challenge model was then used to investigate the efficacy of live attenuated turkey rhinotracheitis ( TRT) vaccine administered alone at 1 day old, or an inactivated TRT vaccine ( at 16 weeks), or a combined programme using both vaccines, in protecting against this challenge. Neither the live nor the inactivated vaccine alone protected against clinical signs ( respiratory infection or diarrhoea). However, the inactivated, but not the live, vaccine did protect against the effect of the i.v. challenge on laying performance. In contrast, the combined vaccination programme protected completely against both clinical signs and poor egg-laying performance. This protection lasted until at least 60 weeks of age. On the basis of the results with this experimental model, it is concluded that the use of live priming followed by administration of inactivated TRT vaccine is necessary to provide complete protection of laying chickens against APV challenge.

Introduction From both field observations and experimental studies, it is well documented that turkey rhinotracheitis ( TRT) virus, otherwise known as avian pneumovirus ( APV), can cause poor egg-laying performance in breeding and laying turkeys ( Stuart, 1989; Cook et al., 1996). The same virus has been isolated from naturally infected chickens ( Picault et al., 1987; Buys et al., 1989; Jones et al., 1991; Gough et al., 1994) and is associated with outbreaks of swollen head syndrome ( SHS) in chickens ( O’Brien, 1985; Hafez, 1993). An antibody response to APV has been demonstrated in chickens ( Wyeth et al., 1987), although not always associated with any disease condition ( Cook et al., 1988). Jones et al. ( 1987) were able to induce mild respiratory disease in young chickens experimen-

tally inoculated with APV and to demonstrate replication of the virus in the respiratory tract by immunofluorescence ( IF). The reproductive tract was not examined in that study. Majo´ et al. ( 1995) demonstrated APV antigen by IF and immunoperoxidase ( IP) assays in respiratory tract tissues of 27-week-old broiler breeders, experimentally inoculated by the oculonasal ( o.n.) route, but failed to demonstrate APV antigen in the oviduct. Catelli et al. ( 1998) used 2-week-old specified pathogen free ( SPF) chickens in a sequential virological and immunochemical study of an experimental APV infection. While virus could be detected by both methods in the upper respiratory tract, it could not be recovered from the oviducts of these young chickens, nor could APV antigen be demonstrated in them by IP. Despite this lack of experimental data to support the role of APV as a pathogen in laying

* To whom correspondence should be addressed. Received 14 March 2000. Accepted 8 June 2000. ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/00/060545-12 © 2000 Houghton Trust Ltd DOI: 10.1080/03079450020016788

546 J. K. A. Cook et al.

chickens, there is increasing evidence that this virus can play a role in SHS and in egg production abnormalities in such chickens in the field ( O’Brien, 1985; Wyeth et al., 1987; Hafez, 1993). In order to be able to confirm the role of APV as a pathogen in chickens, the ability of the virus to cause an effect on egg production needs to be demonstrated under experimental conditions. The objective of the work reported in this paper was twofold. First, to attempt to induce poor egglaying performance by inoculating SPF chickens with APV under experimental conditions, then to study aspects of the pathogenesis of the oviduct infection using an IP technique. Second, to attempt to prevent the effects of the APV infection in laying chickens by vaccination with live-attenuated and inactivated TRT vaccines. Materials and Methods Experimental design Three experiments were performed. Experiment 1: attempt to induce a drop in egg production following APV inoculation. Four groups of 24 hens were used. Group 1 was inoculated o.n. with APV at 37 weeks of age so that each hen received a challenge of log10 6.8 median ciliostatic doses (CD50) of virus in 1 ml. Group 2, which comprised age-matched siblings, was not inoculated and served as the controls for group 1. Group 3 was inoculated with APV by the intravenous (i.v.) route at 49 weeks of age, each hen receiving 1 ml containing log10 5.5 CD50 virus. Group 4 was similarly inoculated i.v. at 49 weeks with Eagle’s minimal essential medium, without serum (MEM), as a placebo. The hens were examined daily and assessed for general condition and for signs of respiratory infection. Egg production and eggshell quality were recorded from 4 weeks before inoculation until 5 weeks post-inoculation (p.i.), when the experiment was terminated. A blood sample was collected from each hen prior to inoculation and when the experiment was terminated. The sera were tested for APV antibodies by enzyme-linked immunosorbent assay (ELISA). Experiment 2: ability of TRT vaccines to protect against the effects of APV challenge on egg laying performance. The experimental design is summarized in Table 1. Groups of 30 hens were used. Group 1 was vaccinated o.n. with live-attenuated TRT vaccine at 1 day old, group 2 received only inactivated vaccine at 16 weeks, while group 3 received both live and inactivated vaccines. These three groups, together with an unvaccinated group (group 4), were challenged i.v. with APV at 28

Table 1. Design of experiment performed to demonstrate the ability of TRT vaccines to protect laying hens against the effects of APV 11/94 on egg-laying performance

Group number

Live priming (1 day old)

Inactivated vaccine (16 weeks)

Challenge (28 weeks)

weeks of age, each hen receiving log10 6.4 CD50 virus in 1 ml. Group 5 (fully vaccinated) was inoculated i.v. with 1 ml MEM as a placebo. Egg production and eggshell quality were recorded from 3 weeks pre-challenge to 5 weeks post-challenge, when the experiment was terminated. Blood samples were collected from all hens at intervals throughout. Experiment 3: duration of immunity. A group of 30 hens that had been given the full TRT vaccination programme of both the live-attenuated and inactivated TRT vaccines, as already detailed (experiment 2), and a group of 15 hens that had received no TRT vaccine were reared until 60 weeks of age. They were then challenged i.v. as already described with a dose of log10 6.4 CD50 virus. Egg production and eggshell quality continued to be monitored until 5 weeks post-challenge, when the experiment was terminated. Chickens SPF chickens were used in all experiments. They were vaccinated against Marek’s disease at 1 day of age, but received no further vaccinations other than those detailed in each experiment. They were housed under negative pressure in sheds with a filtered air supply. The males were culled as soon as they could be identified. The chickens were reared on litter, then the females transferred to individual laying cages prior to onset of lay. To minimize the risk of spread of infection between houses, separate protective clothing was provided for each house and uninoculated birds were always visited first. TRT vaccines Live-attenuated vaccine. A commercially available vaccine, based on a subgroup A strain, was used (TRT vaccine Nobilis, Intervet International, Boxmeer, The Netherlands). It was administered o.n. at 1 day old so that each chick received one manufacturer’s recommended dose of vaccine. Oil-adjuvanted inactivated TRT vaccine. An experimental vaccine was used, containing, in addition to TRT antigen: Newcastle disease, infectious bursal disease, EDS–76 and infectious bronchitis (M41 & D274) antigens. The vaccine was formulated to contain the manufacturer’s normal amounts of each antigen. It was administered (0.5 ml) into the gastrocnemius muscle at 16 weeks of age. APV challenge virus The challenge strain used, designated 11/94 (subgroup B), has been described previously (Cook et al., 1995; Catelli et al., 1998). It had received five to seven passages in tracheal organ cultures (TOC) before use in these experiments. It was assayed in TOC (Cook et al., 1976) and the titre calculated by the method of Reed & Muench (1938). Histology and immunostaining Samples of tissue from the following organs were taken for histological examination: sinus/turbinate, trachea, lung, liver, kidney, intestinal tract (five levels), oviduct (four levels) and shell gland. They were fixed in modified Bouin’s fixative (J¨onsson & Engstr¨om, 1986), processed by conventional methods and embedded in paraffin wax. They were then stained by haematoxylin and eosin (H&E) and by peroxidase antiperoxidase (PAP) immunostaining for TRT antigen, using a normal mouse serum and a mouse anti-TRT monoclonal antibody as described previously (Catelli et al., 1998). Egg production and eggshell quality

1 2 3 4 5 a

Yes No Yes No Yes

No Yes Yes No Yes

Yes Yes Yes Yes Placeboa

Eagle’s minimum essential medium without serum.

In each experiment, the daily egg production of each hen was recorded individually. The percentage egg production for each group was calculated on a weekly basis: 100% egg production being equivalent to each hen laying one egg on each day. All eggs laid were also candled and the following egg shell faults recorded: crack, hair crack, thin, soft, ridged, misshapen shells, blood spot, meat spot. The incidence of poor eggshell quality was calculated as a percentage of all eggs laid/group/ week.

Avian pneumovirus in laying hens 547 Serology Sera were tested for APV antibodies by ELISA as described previously (Cook et al., 1996). Statistical analysis The egg production data were analyzed by the analysis of variance method using a SAS programme. The eggshell quality data were analyzed using a chi-squared test, applying continuity correction for the 2 x 2 tables.

Results Experiment 1 Clinical signs following APV inoculation. Hens were not examined individually for signs of respiratory infection because of the risk that handling them might adversely affect egg production. Instead, the group as a whole was observed each day. Coughing was heard in a few hens in group 1 between 4 and 6 days p.i., and in a few hens in group 3 between 2 and 8 days p.i. No coughing was heard in the two control groups at any time. Severe diarrhoea was observed in some birds inoculated with APV by each route. Following o.n. inoculation, up to five hens had diarrhoea between 17 and 30 days p.i. Following intravenous inoculation, diarrhoea was observed in over one-half of the hens between 4 and 16 days p.i. Some hens in this group had diarrhoea on several consecutive days, while others were never affected. No other signs of

infection were seen in the group that was inoculated o.n. However, following i.v. inoculation, approximately 70% of the hens appeared very subdued, and some looked sick between 4 and 11 days p.i. No sound was heard in the shed and the hens sat quietly with their feathers ruffled. Mortality following APV inoculation. There was no mortality in either of the control groups or in the group that was inoculated o.n. One hen died 6 days after i.v. inoculation. Postmortem examination showed that the respiratory tract was normal but revealed the presence of severe egg peritonitis ( Table 2). Pieces of tissue from four different parts of the oviduct were taken for histological examination. Histological examination of oviduct tissue. The hens in the group inoculated i.v. that were sick or moribund between 7 and 11 days p.i., were killed humanely. Two were killed at 7, 8, and 9 days p.i., and one sick hen plus one healthy one ( killed as a ‘control’) at 11 days p.i. One moribund hen was killed at 32 days p.i. The results of the postmortem examinations are summarized in Table 2. Mucus was present in the trachea of one hen killed at 7 days p.i., and slight nasal exudate was seen in one hen killed at 9 days p.i.; otherwise, the respiratory tract of all hens appeared normal. The only abnormalities observed were in the reproductive

Table 2. Postmortem, histological and immunochemical examination of hens that were killed following intravenous inoculation of APV 11/94 ( experiment 1) Histological examination of oviduct epitheliuma Diarrhoea

Clinical condition

Postmortem findings

H&E

IPb

6

Severe

Dead

Severe egg peritonitis

Some epithelium still present; detached in places

++ c

7

Severe Severe

Moribund Sick

Mucus in trachea; egg peritonitis Egg peritonitis; haemorrhage on ovum

Damaged; some sloughing off Damaged but largely intact

–

8

Severe Severe

Sick Sick

Egg peritonitis Haemorrhages on ova

90% damaged or absent Mostly absent

++ ++ d

9

Some

Sick Sick

Some damage, but largely intact; loss of cell density Largely intact

++ e

None

Slight nasal exudate; inspissated yolk in shell gland Inspissated yolk in shell gland

Severe

Sick

Largely intact; some loss of cell density

–

None

Good

Egg peritonitis; inspissated yolk in shell gland Normal

Normal

–

None

Moribund

Ovary and oviduct regressed

Epithelium intact; normal

–

Day p.i.

11

32 a

All tissues except oviduct were normal. ++ , APV antigen detected strongly; + , some APV antigen detected; –, negative. c See Figure 1. d APV antigen also seen in the debris in the lumen of the oviduct. e See Figure 2. b

++

+

548 J. K. A. Cook et al.

(a)

( b) Figure 1. Shell gland of a hen that died 6 days after intravenous challenge with APV at 49 weeks of age. ( a) Stained with APV monoclonal antibody. (b) Stained with normal mouse serum. Bar = 12.5 mm.

tract, where egg peritonitis was generally observed. The ovary and oviduct of the moribund hen killed at 32 days p.i. had regressed, but no APV antigen was detected after IP staining. Tissues were also examined after H&E and PAP staining. The only abnormalities seen

histologically were in the oviduct. In most cases, the epithelium was absent or damaged and APV antigen was detected by IP staining in the hens that were examined up to 9 days p.i. Examples of these results are shown in Figures 1 and 2.

Avian pneumovirus in laying hens 549

Figure 2. Shell gland of a hen killed 9 days after intravenous challenge with APV at 49 weeks of age. Stained with APV monoclonal antibody. Bar = 12.5 mm.

Egg production and eggshell quality. The egg production and incidence of poor eggshell quality recorded following o.n. inoculation of APV is shown in Figure 3a. The results clearly show that inoculation of APV by this route did not affect either the numbers of eggs laid or the shell quality. The egg production following i.v. inoculation is shown in Figure 3b. Because one hen died in this group and other sick hens were humanely killed at different times following inoculation, the egg production data for only the 15 hens that remained until the end of the experiment is shown. Despite this rather small number of hens, it is clear that i.v. inoculation of APV 11/94 severely affected egg production. Egg numbers had fallen within 1 week of inoculation and, by 2 weeks p.i., egg production had dropped to 26.7%. Egg numbers increased again over the next 2 weeks, but had barely returned to the pre-inoculation level when the experiment was terminated 5 weeks post-challenge. The incidence of eggshell faults recorded following i.v. inoculation of APV 11/94 is shown in Figure 3c. The higher level of eggshell faults recorded prechallenge in the inoculated group can be accounted for by two hens consistently laying cracked eggs. Following i.v. challenge, the incidence of poor shell quality increased sharply in that group. This poor shell quality was mainly accounted for by a large increase in the numbers of soft or thin-shelled eggs, particularly as hens that had stopped laying came back into lay. No soft-shelled eggs were laid by this

group pre-challenge but, in the 4 weeks p.i., 7, 18, 27 and 8%, respectively, of all eggs laid were softshelled. Eggshell quality improved somewhat as the egg production increased again, but poor-quality eggs continued to be laid until the end of the experiment. Examination of hens postmortem. The experiment was terminated 5 weeks p.i. and the surviving hens examined postmortem. The oviduct of one hen, which was still out of lay, was severely regressed, and inspissated egg yolk was found in the peritoneum. The oviducts of all the other hens appeared normal. When oviduct tissue was examined histologically, no damage was observed and no APV antigen was detected using the PAP assay. Serology. Sera collected pre-challenge and when the experiment was terminated were tested for APV antibodies by ELISA. All pre-inoculation sera were negative, as were those from the two control groups. Both APV-inoculated groups showed a strong antibody response to APV challenge, mean titres in both groups being >log2 16. Experiment 2 Clinical signs following APV inoculation. The clinical signs seen following i.v. challenge of groups of hens given different TRT vaccination programmes, or not vaccinated ( Table 1), are summarized in Table 3. In order to minimize the

550 J. K. A. Cook et al.

(a)

( b)

(c) Figure 3. (a) Egg production ( top) and eggshell quality (bottom) following oculonasal inoculation of 37-week-old SPF hens with APV 11/94. (b) Egg production following intravenous inoculation of 49-week-old SPF hens with APV 11/94. ( c) Eggshell quality following intravenous inoculation of 49-week-old SPF hens with APV 11/94. Filled diamonds, inoculated; filled circles, control.

Avian pneumovirus in laying hens 551

(a)

( b)

(c) Figure 4. ( a) Egg production before and after intravenous APV challenge at 28 weeks of TRT-vaccinated and unvaccinated hens. ( b) Eggshell quality before and after intravenous APV challenge at 28 weeks of TRT-vaccinated and unvaccinated hens. ( c) Egg production in groups of TRT-vaccinated and unvaccinated hens before and after intravenous APV challenge at 60 weeks of age. Diamonds, solid line, Unvaccinated; circles, solid line, live only; diamonds, dotted line, inactivated only; circles, dotted line, live, inactivated; dashed line, unchallenged.

552 J. K. A. Cook et al. Table 3. Clinical signs following inoculation of 28-week-old vaccinated or unvaccinated hens intravenously with APV11/94 (experiment 2) Group 1 Days p.i.

Ca

TRT vaccine

TRT challenge 4 5 6 7 8 9 10 12 14

– – +

– – – –

Sb

Group 2 Dc

C

S

Group 3 D

C

S

Group 4 D

C

S

Group 5 D

C

S

Live priming only

Inactivated only

Live, inactivated

None

Live, inactivated

Yes

Yes

Yes

Yes

No

0 0 1 NDd 4 ND 3 1 0

0 0 1

–

4

– – – – –

1 1 0

+ + +

0 0 2 1 3 0 0 0 0

0 0 0 1 2 0 0 1 0

– – – – – – –

0 0 0 ND 0 ND 0 0 0

0 0 0 0 0 0 0

– + ++ +

– – – – –

1 2 6 15 10 3 1 1 0

1 1 2 9 13 0 1 1 0

– – – – – –

0 ND 0 ND 0 ND 0 0 0

D

0 0 0 0 0 0

a

Coughing: ++ , moderate; + , slight; –, no coughing. Number of hens that appeared sick c Number of hens with diarrhoea. d ND, group not examined on that day. b

risk of spreading APV infection among the groups, not every group was examined on every day; instead, only the groups of particular interest were examined. Coughing was heard in the unvaccinated, challenge control group between 5 and 7 days p.i., and to a lesser extent in the groups given only either live or inactivated vaccine. Almost 50% of the unvaccinated, challenge control hens were sick and/ or had diarrhoea at some time following challenge. Although the hens in this experiment did not appear to be as unwell as the hens in experiment 1, sick hens, with or without diarrhoea, were seen in this group between 4 and 12 days p.i. A smaller number of sick birds and a lower incidence of diarrhoea were also recorded in the groups that were given only either the live or the inactivated TRT vaccine. No sick hens were seen in the fully vaccinated, challenged group or in the vaccinated, unchallenged group. Mortality following APV challenge. One hen died at 26 weeks of age in the vaccinated, unchallenged group; it had a prolapsed oviduct. Egg production and eggshell quality. The numbers of eggs laid between 3 weeks pre-challenge and 5 weeks post-challenge are shown in Figure 4a. There were no significant differences in egg production between groups prior to challenge. As in experiment 1, i.v. challenge caused a substantial drop in egg production in the unvaccinated hens. At 2 weeks post-challenge, production had fallen to 25%; thereafter it recovered, but had not returned to

the pre-challenge level by the time the experiment was terminated 5 weeks post-challenge. The egg production in this group was significantly different from that in the unchallenged group and also in each vaccinated group ( P < 0.001). Prior vaccination with a combination of live-attenuated and inactivated TRT vaccines protected the hens completely against the effect of challenge on egg production ( P = 0.3). Inactivated vaccine, given without live priming, also protected very well against the effects of APV on egg production. However, the live priming alone protected poorly. During the first 2 weeks after challenge, the egg production in the group given the live vaccine alone was significantly lower than in the group given the full vaccination programme ( P < 0.001). The incidence of poor eggshell quality is shown in Figure 4b. Although some shell faults were seen in all groups prior to challenge, differences between groups were not sufficiently large to prevent analysis of the post-challenge data by the chisquared test. A marked increase in eggshell faults was recorded in the unvaccinated, challenge control group ( P < 0.001, compared with the unchallenged group). Again, this was mainly a result of an increased incidence of soft or thin shelled eggs. Live-attenuated TRT vaccine given alone provided only very poor protection against the effects of challenge on eggshell quality. Compared with the unchallenged group, the difference was highly significant ( P < 0.001). The inactivated vaccine given alone protected well, with no significant difference from the unchallenged group ( P = 0.54).

Avian pneumovirus in laying hens 553 Table 4. APV antibody response ( log2 ) of groups of 30 hens to TRT vaccination followed by intravenous challenge at 28 weeks of age ( experiment 2) TRT vaccine TRT challenge Time bled

Live priming only

Inactivated only

Yes

Yes a

Live, inactivated

None

Yes a

Live, inactivated

Yes a

No a

APV antibody titre APV antibody titre APV antibody titre APV antibody titre APV antibody titrea

Pre-inactivated vaccine ( 16 weeks)

8.6 ( 1.8)

£7.0 ( 0.6)

9.4 ( 2.3)

Pre-challenge(28 weeks)

8.1 ( 1.7)

11.4 (1.7)

³15.2 ( 2.5)