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Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20

The use of virus isolation, histopathology and


immunoperoxidase techniques to study the
dissemination of a chicken isolate of avian
pneumovirus in chickens

E. Catelli , J. K. A. Cook , J. Chesher , S. J. Orbell , M. A. Woods , W. Baxendale &


M. B. Huggins

To cite this article: E. Catelli , J. K. A. Cook , J. Chesher , S. J. Orbell , M. A. Woods ,


W. Baxendale & M. B. Huggins (1998) The use of virus isolation, histopathology and
immunoperoxidase techniques to study the dissemination of a chicken isolate of avian pneumovirus
in chickens, Avian Pathology, 27:6, 632-640, DOI: 10.1080/03079459808419395

To link to this article: https://doi.org/10.1080/03079459808419395

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Avian Pathology (1998) 27, 632-640

The use of virus isolation, histopathology and


immunoperoxidase techniques to study the
dissemination of a chicken isolate of avian
pneumovirus in chickens

E. Catelli1*, J. K. A. Cook2, J. Chesher2, S. J. Orbell2, M. A. Woods2, W.


Baxendale2 & M. B. Huggins2
1
Dipartimento di Sanità Pubblica Veterinaria e Patología Animale—Sezione di Patología Aviare,
Facoltà di Medicina Veterinaria, Université di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia
(BO), Italy and 2Intervet UK Ltd, The Elms, Thicket Road, Houghton, Huntingdon, Cambs. PE17 2BQ,
UK

A subgroup B isolate of turkey rhinotracheitis virus (TRTV) or avian pneumovirus (APV), obtained from
a flock of commercial breeding chickens experiencing poor egg production, mortality and swollen head
syndrome, was shown to cause substantial respiratory signs in both young SPF chickens and chicks with
high levels of maternally derived TRT antibodies.
This isolate replicated to high titre in the respiratory tract of experimentally inoculated SPF chickens
for approximately 5 days after inoculation, but was recovered only occasionally after that time. It was
never recovered from non-respiratory tract tissues. A detailed, sequential histological and immunoperox-
idase study was performed. This revealed that, whilst TRT virus could be demonstrated consistently in the
epithelium of upper respiratory tract tissue, although only for a short time after inoculation, the damage
which it caused was minimal and recovery was rapid. This study, using a pathogenic TRT isolate obtained
from diseased chickens, provides clear evidence that TRT virus can cause damage to the respiratory tract
of chickens and that this damage is both localized and short lived.

Introduction there might be biological differences in the in vivo


behaviour of TRT viruses isolated from the two
Turkey rhinotracheitis (TRT) was first reported in
species, although these differences were not related
South Africa in the late 1970s (Buys & du Preez,
to particular subgroups of the virus.
1980) and the causal agent subsequently character-
Virus dissemination studies have been per-
ised as a pneumovirus (Cavanagh & Barrett, 1988).
formed with TRT virus in turkeys (Cook et al,
Only one serotype of TRT virus has so far been
1991). The presence of the virus in turkey tissues
described (Cook et al, 1993a), but on the basis of
has been reported by Jones et al. (1986, 1988)
differences in the sequence of the G glycoprotein,
using immunofluorescence (IF) and by O'Loan &
two subgroups (A & B) within that serotype have
Allan (1990) using immunoperoxidase (IP) assays.
been reported (Juhasz & Easton, 1994). TRT virus
Majó et al. (1995) used IP, and Jones et al. (1987)
is the cause of a well defined disease in turkeys
used virus isolation, histology and IF techniques to
and can also infect chickens (Cook et al, 1988),
study the ability of a turkey isolate of TRT virus to
although its role as a pathogen in that species is
infect chickens. Cook et al. (1993b) compared the
still poorly understood. TRT viruses have been
infection caused by South African TRT isolates
isolated from chickens (Picault et al, 1987; Buys
obtained from turkeys and chickens in the two
et al, 1989; Jones et al, 1991), and Jones et al.
species. However, the pathogenesis of TRT virus
(1987) were able to infect chickens with the virus,
in the chicken requires further elucidation.
but without causing apparent disease. Buys et al.
(1989) and Cook et al (1993b) have suggested that In 1994, we obtained an isolate of TRT virus

*To whom correspondence should be addressed.


Received 29 January 1998. Accepted 22 April 1998.

0307-9457/98/060632-09 © 1998 Houghton Trust Ltd


Sequential pathogenesis study of TRT in chickens 633

from commercial breeding chickens experiencing Virus re-isolation


some mortality, poor egg production and signs of For all tissues except duodenum, 10% w/v tissue homogenates were
swollen head syndrome. The isolate was shown to prepared in Eagle's minimal essential medium without serum (SF). In
be a subgroup B strain of TRT and was designated the case of duodenum, a 50-mm length of the organ was opened
TRT 11/94 (Cook et al, 1995). Preliminary indica- longitudinally and the epithelial lining scraped, using a scalpel blade,
into a weighed bottle. SF was then added to give a 10% w/v
tions were that this virus was pathogenic for chick- suspension. All samples were examined for virus content in OC as
ens, and this paper reports experiments performed described previously (Cook et al, 1991), ciliostasis being taken as the
to study aspects of the pathogenesis of this virus in indicator of the presence of virus. For respiratory tract tissues, a series
the chicken and describes a sequential study under- of 10-fold dilutions was inoculated and the titrations were examined
taken to determine the tissue distribution and per- for absence of ciliary activity (ciliostasis) 7 days later. When ciliosta-
sis was not observed and in the case of all other tissues, undiluted
sistence in that species, using virus isolation, samples were given three blind passages in OC at 3 to 4-day intervals.
histological and immunochemical methods. The third passage was incubated for 7 to 10 days, then OCs were
examined for ciliostasis.

Materials and Methods


Histology
Virus strain
Tissue samples were fixed in a modified Bouin's fixative (Jönsson &
The subgroup B TRT virus isolate, designated 11/94 (Cook et al,
Engström, 1986), processed by conventional methods and embedded
1995), was originally isolated in chicken embryo trachéal organ
in paraffin wax. Consecutive sections were stained by haematoxylin
cultures (OC) and had received four (Experiment 1 and 2) or seven
and eosin (H&E) and by peroxidase anti-peroxidase (PAP) immunos-
(Experiment 3) passages in OC prior to use. It was used here as OC
taining for TRT antigen, using a mouse anti-TRT monoclonal anti-
supernatant fluid and was assayed in OC (Cook et al, 1976). The dose
body which recognises the G glycoprotein.
was logio 5.6 median ciliostatic doses (CD5o)/chick (Experiment 1),
logio 3.5 CD50/chick (Experiment 2) and logio 5.0 CDso/chick (Exper-
iment 3).
Peroxidase anti-peroxidase immunostaining
Tissue sections were dewaxed in two changes of xylene and passed
Chickens through two changes of industrial methylated spirit (IMS). Endoge-
nous peroxidase was blocked using 0.3% H2O2 in IMS for 30 min at
Specified pathogen-free chicks. Eggs were obtained from a commer-
room temperature. Following two 15-min washes in Tris buffered
cial specified pathogen free (SPF) flock, then incubated and hatched at
saline solution (TBS), tissues were incubated with a blocking agent,
Intervet UK.
normal goat serum (1:40 dilution) for 1 h at room temperature.
Commercial chicks. Chicks were obtained at 1-day-old from a com- Excess blocking serum was removed, the section covered with a TRT
mercial broiler chick hatchery. They were the progeny of hens given monoclonal antibody (1:1000 dilution) and incubated overnight in a
both live-attenuated and oil-adjuvanted inactivated TRT vaccines. humidified chamber at + 4°C. After two 15-min washes in TBS,
tissues were covered with goat anti mouse serum (1:40 dilution) for
1 h at room temperature. Following two further washes in TBS, the
Experimental design sections were covered with mouse PAP (1:100 dilution) for 1 h at
room temperature. Tissues were further washed in TBS followed by
Virulence studies. Three- or 24-day-old SPF chickens (Experiment 1) acetate/citrate buffer (pH 6) then placed in nickel enhanced DAB
or 1-day-old commercial chicks with maternally derived TRT anti- substrate as described by Shu et al. (1988) for 4 min. After three
bodies (Experiment 2), housed in negative pressure isolators were 5-min washes in TBS to stop the reaction, the sections were counter-
used. They were inoculated by the oculonasal route (100 ¿il per chick), stained for 6 s with 0.5% Light Green stain, dehydrated in IMS,
then examined for clinical signs of infection up to 10 days post-inoc- cleared in xylene and mounted. Known TRT-positive sections were
ulation (d.p.i.). Each chick was examined individually and the clinical included in the staining procedure.
signs scored as follows: All the reagents were obtained from SIGMA Chemicals Poole,
Dorset, UK.
1. Nasal exúdate when nares gently pressed
2. Obvious nasal exúdate
3. Copious nasal exúdate
Serology
4. Turbid nasal exúdate
5. Ocular discharge Sera were tested for TRT antibodies by ELISA as described previ-
6. Swollen sinus ously (Cook et al, 1996).

Virus dissemination and histological study (Experiment 3). Three-


week-old SPF chickens housed in negative pressure isolators were
used. They were inoculated with a total of 100 n\ of TRT virus. This
was divided into four equal parts, so that 25 ß was applied into each Results
nostril and 25 ß\ onto each eye. Nine siblings were bled. Two chicks
were killed at 1, 2, 3, 4, 5, 6, 7, 10, 14, 18 and 21 d.p.i. The following Clinical signs following challenge with TRTV 11/
tissues were removed aseptically in the following order: Liver*, 94
airsact, ovary/testist, kidney*, bursa of Fabricius*, duodenum*, cae-
cal tonsil*, lung*, trachea*, sinus tissue*, corneat. Tissues marked * The clinical signs recorded in Experiments 1 and 2
were taken for attempted virus recovery and histological examination; are summarized in Figure 1, where the clinical sign
those marked t were only examined histologically. One nasal tur- scores on different days p.i. are shown. The only
binate was examined histologically, the remaining nasal tissue was signs recorded were nasal exúdate, which was
taken for attempted virus recovery. In the case of trachea, the upper
sometimes copious and/or turbid. No ocular dis-
portion was used for virology and a portion of the lower part fixed for
histological examination. charge or swollen sinuses were observed. The
Twenty chicks were bled 14 d.p.i. and the sera tested for TRT extent and severity of the signs observed were
antibodies by ELISA. similar whether SPF or commercial chicks with
634 E. Catelli et al.

Mean clinical sign score / chick summarized in Table 1. Virus was recovered in
large amounts from nasal tissue, sinus tissue and
trachea of all chicks examined up to 5 d.p.i. Virus
was also recovered, but in smaller quantities, from
most, but not all, samples of lung from one to 5
d.p.i. Thereafter, only small quantities of virus
were recovered from the respiratory tract and only
some chicks were positive. Virus was rarely found
after 6 d.p.i. and none was recovered at 18 or 21
d.p.i.
No TRT virus was recovered from the kidney,
liver, duodenal scrapings, bursa of Fabricius or
caecal tonsils of any of the chickens at any time
during the study.

4 5 6 10 Immunoperoxidase staining
Day post inoculation
The results of using IP staining to detect TRT
antigen in tissues are summarized in Table 2. TRT
• 3-day-old SPF chicks antigen was demonstrated consistently in nasal
turbinate between 1 and 5 d.p.i. (Figures 3, 5b, 6b
1124-day-old SPF chicks and 7b), where it was confined to the ciliated
• 1-day-old maternal antibody positive chicks border of the respiratory epithelium (Figure 3).
Three and four d.p.i., the specific IP staining was
Figure 1. Clinical sign scores in chickens inoculated with a found extensively in the epithelial cells and also in
subgroup B strain of TRT virus by the oculonasal route. the catarrhal exúdate (Figure 6b). A few scattered
cells also showed specific cytoplasmic staining.
TRT antigen was not detected in nasal turbinate
maternally derived TRT antibodies were used, tissue from 6 d.p.i. (Figure 8b). Antigen was
although their onset was delayed slightly in the detected in the trachea of one bird killed 4 d.p.i. It
commercial chicks. was also detected in both the sinus (Figure 12c)
and trachea (Figure lid) of one of the two birds
killed 5 d.p.i. It was not detected in any other
Re-isolation of TRT vims from tissue samples tissue on any occasion during the study. The dark
The results of attempted virus recovery from res- staining in small areas in the lamina propria has to
piratory tract tissues of chicks in Experiment 3 are be regarded as non-specific as such areas are

Table 1. Recovery of TRT virus from the respiratory tract of chickens inoculated with
a pathogenic subgroup B strain by the oculonasal route

Amount of virus recovered1 (log10 CD50)


' Per g of tissue :from

Day post- Nasal Sinus


inoculation tissue tissue Trachea Lung

1 5.7 6.4a 5.5 5.4 3.2 4.5 3.3 ; b

2 5.4 7.2 5.0 6.7 5.2 4.8 2.8 2.3


3 6.2 7.5 4.8 5.8 4.8 5.3 2.6 2.5
4 5.8 7.7 5.3 6.5 6.4 6.2 3.2 2.6
5 7.4 5.5 4.5 3.8 4.3 5.0 — 2.5
6 2.5 — 2.3 — — — 2.2 2.4
7 2.0 — 2.2 — — — — —
10 — — 2.3 — — — — —
14 — 2.0 — — — — — —
18 — — — — — — — —
21 — — — — — — — —
a
Two chicks were killed on each occasion.
b
No virus detected after three passages in OC; minimum amount of virus detectable was
log io 2.0 CD5o per g of tissue.
Sequential pathogenesis study of TRT in chickens 635

Table 2. Results of examining tissues of chickens killed sequentially after


inoculation with a virulent strain of TRT virus, 11/94, for presence of TRTV antigen
using an immunoperoxidase assay

Incidence of TRTV antigen in tissues


at different times after inoculation

Day pi Nasal turb. Sinus Trachea

1
2
3
4
5
6
7
10
14
18
21
a
+ , TRTV antigen present.
b
± , TRTV antigen probably present.
c
+ + , TRTV antigen detected strongly.
d
—, Negative for TRTV antigen.
All other tissues examined were negative for TRTV antigen.

occasionally seen in tissues from uninfected birds increased so that the mucus glands appeared hyper-
where there is an accumulation of inflammatory trophic (Figure 9a). This hypertrophy lasted at least
cells and blood vessels. until 21 d.p.i. Ten and 14 d.p.i., areas of the lamina
propria were still heavily infiltrated by
lymphocytes, sometimes aggregated in lymphoid
Histopathological study nodules, and were covered by abnormal flattened
Microscopical changes were seen only in the res- epithelial cells devoid of cilia (Figure 9b). Recov-
piratory tract. The most significant lesions were ery was complete by 18 d.p.i. (Figure 10).
observed in the nasal turbinâtes between 3 and 10
d.p.i. The histological changes seen in the nasal
turbinate were almost always seen in the internal Trachea. Thickening of the mucosa, due to conges-
side of the turbinate spiral and the entire mucosa tion, oedema and mononuclear cell infiltration in
was never affected. Sinuses and tracheas were also the lamina propria was observed 4 and 5 d.p.i. This
affected, but the tissue damage and the was associated with flattening of the epithelial cells
inflammatory changes were less evident and lasted and focal deciliation (Figure lie). Subsequently,
for a shorter time. only focal deciliation of the ciliated epithelium,
with occasional mononuclear cellular infiltration in
the lamina propria, was seen up to 7 d.p.i.
Nasal tissue. One and 2 d.p.i only mild mononu-
clear cell infiltration and oedema were seen in the
lamina propria (Figure 4). From 3 d.p.i., the pseu- Sinus. From 4 to 10 d.p.i., the mucosa lining the
dostratified ciliated columnar epithelium became infra-orbital sinus showed hyperplasia and focal
swollen and vacuolated and mucous exúdate pro- exfoliation of the columnar ciliated epithelium.
duction was increased, as was the inflammatory The changes in the epithelium were accompanied
cellular infiltration and hyperaemia of the lamina by congestion, oedema, haemorrhages and
propria (Figure 5a). From 4 d.p.i., epithelial cell mononuclear cellular infiltration in the lamina pro-
exfoliation and catarrhal exúdate were also seen in pria (Figure 12a,b), together with mucous exúdate
the lumen (Figure 6a). Five d.p.i., small areas in the lumen. Subsequently, only focal deciliation,
showed sloughing off of the epithelium (Figure with occasional mononuclear cellular infiltration,
7a). Six and 7 d.p.i. some limited areas of the was seen up to 18 d.p.i.
lamina propria, thickened because of mononuclear
infiltration and hyperaemia, were covered by a
single thin epithelial layer that still appeared to be Other tissues. No histopathological changes were
ciliated (Figure 8a). Glandular secretion was observed in any of the other tissue examined.
636 E. Catelli et al.

Serology
All SPF chicks were free from TRT antibodies
(titres < Iog2 4,6) prior to use in these experiments
(data not shown). Siblings of the commercial
chickens used in Experiment 2, bled at the time of
inoculation, had TRT antibody titres ranging from
log2 10.0 to >log2 13, with a mean titre of Iog2
12.7.
In Experiment 3, 20 chicks were bled 14 d.p.i.
All had TRT antibody titres of >log 2 13.

Discussion
The results of the challenge experiments reported
here and the clinical reports from the farm from
which this isolate derived, suggest that TRTV
11/94 is a particularly virulent strain for chickens.
It was also interesting to note that the clinical signs
recorded in the 1-day-old commercial chicks with
high levels of maternally derived TRT antibodies
were as severe as those recorded in the SPF chicks,
confirming earlier findings in turkeys (Cook et al,
1989) which showed that poults could be infected
with TRT virus in the face of high levels of
maternally derived TRT antibodies, with no obvi-
ous modification of the clinical signs. Jones et al.
(1987) failed to detect TRT antigen by IF or to
recover virus from the trachea of experimentally
inoculated 3-week-old commercial broiler chick-
ens. The different results of the two studies may
possibly be explained by the use of different chal-
lenge strains; a turkey isolate being used in the
earlier study whilst a virulent chicken isolate was
used here.
In this study, very large amounts of TRTV 11/94
were recovered from the respiratory tract of SPF
chickens for a few days after inoculation, but the
virus was cleared very quickly and was recovered
only occasionally beyond 5 d.p.i. Previous studies
(Cook et al., 1991) have shown similar replication
of a turkey isolate of TRT virus in the respiratory
tract of turkeys. Surprisingly, given this high virus
replication in the respiratory tract, virus was never Figure 2. Nasal turbinate from a 3-week-old uninoculated
recovered from any other sites. However, these chicken, (a) stained with H&E, (b) PAP immunostaining for
TRTV antigen, Light Green counter stain. Same area as (a),
findings do confirm earlier studies, all of which
consecutive section. (Bar = 25 um).
indicate that TRT virus is very much a pathogen of Figure 3. Nasal mucosa of a chicken inoculated with a
the respiratory tract (Jones et al., 1988; Cook et al, subgroup B strain ofTRT virus (11/94). Two d.p.i., TRTV antigen
1991, 1993b; Majó et al, 1995). (dark staining) confined to the ciliated border of the respiratory
Cook et al. (1993b) compared the replication of epithelium. (PAP immuno stain for TRTVantigen. Bar= 10 ¡xm).
both turkey and chicken isolates of TRT virus in Figure 4. Nasal turbinate 2 d.p.i., showing oedema and mild
both species and suggested that a TRT isolate mononuclear cellular infiltration in the lamina propria (H&E,
derived from chickens replicated to higher titre in bar = 25 \im).
the respiratory tract of that species. However, the
virus titres reported here are higher than those
found in the previous study. The use in the present
study of a virulent TRT isolate from chickens may that tissue. However, it was surprising that the IP
explain the differences in the results of the two assay detected TRT antigen so poorly in other
studies. respiratory tract tissues, particularly in trachea and
The times at which TRT antigen was detected in sinus. Since the sinus and nasal cavity are directly
nasal turbinate tissue by the IP assay correlated connected, it is possible that some of the virus
with the results of virus reisolation attempts from recovered from the sinus was actually produced in
Sequential pathogenesis study of TRT in chickens 637

life.

Figure 5. ¿Vasa/ turbinate 3 d.p.i. (a) hyperaemia and inflammatory cellular infiltration of the lamina propria are increased at this
stage (H&E). (b) Strong TRTV specific staining (PAP immunostain. Same area as (a), consecutive section). Bar = 25 \xm.
Figure 6. Nasal turbinate 4 d.p. i. (a) epithelial cell exfoliation with catarrhal exúdate in the lumen, hyperaemia and heavy inflammatory
cellular infiltration of the lamina propria (H&E). (b) TRTV antigen found extensively in the ciliated epithelium and in the catarrhal
exúdate [PAP immunostain, consecutive section to (a)]. Bar = 25 urn.

the nasal cavity, which could explain the dis- tions. The virus isolation techniques used by differ-
crepancy in TRTV detection methods for the sinus. ent workers may also have contributed to the
IP staining was performed only on thin sections differences. For example, in the present study,
of each tissue sample, whereas much larger pieces samples were passages 3 times in OC before being
of tissue were assayed for virus content and this considered negative.
may, at least in part, explain the different findings. The complete failure to detect TRT antigen in
Furthermore, only the upper portion of the trachea either lung or airsac by the IP assay was surprising,
was used for attempted virus recovery, whilst a given that TRT virus was recovered from lung
piece taken from the lower portion was examined occasionally in the present study and recovery
histologically and this may also have contributed from both lung (Cook et al, 1993b; Majó et al,
to the different results obtained. Majó et al. (1995) 1995) and airsac (Cook et al, 1993b) of TRT-
considered the IP assay to be highly sensitive in infected chickens has been reported previously.
detecting TRTV antigen in respiratory tract tissues, However, as discussed above, this difference might
but they only detected the antigen in the trachea of be explained by the limited amount of tissue exam-
chickens inoculated by intratracheal route as well ined by the IP assay. Also in the case of the lung
as by eyedrop and conjunctival routes. Jones et al. sections, these were taken from the middle portion
(1988) found IF to be more sensitive than virus with no primary bronchi, thus it is possible that the
recovery in their studies with TRT-free turkeys. anterior portion and primary bronchi may have
These workers detected viral antigen in nasal tur- contained detectable infected cells.
binate for up to 5 d.p.i. which is in agreement with The histopathological features observed in the
the present findings in chickens. However, they sinus, nasal tissue and trachea were similar to those
were able to detect TRTV antigen by IF in the previously described in experimentally infected
tracheas of turkeys between 1 and 7 d.p.i. This chickens (Jones et al, 1987; Majö et al, 1995) and
may possibly suggest that IF is more sensitive than were not substantially different from those seen in
IP, but such a conclusion could not be drawn from experimentally-infected turkeys (Jones et al, 1986;
these limited data. This could only be confirmed by Majö et al, 1995). However, in this study, no
a detailed comparison of the two techniques in the heterophil infiltration was observed in the lamina
same species of bird under exactly the same condi- propria (Jones et al, 1986) nor was purulent exu-
638 E. Catelli et al.

•i vJ

J I*' M ' * "- l »" * ab


Figure 7. MzscZ turbinate 5 d.p.i. (a) showing epithelium Figure 9. Nasal turbinate. (a) 7 d.p.i. Hypertrophie mucous
sloughed off (H&E). (b) TRTV antigen in the epithelium and glands and catarrhal exúdate in lumen, (b) 10 d.p.i. Limited
detached cells (PAP immunostain, consecutive section). areas of the lamina propria still heavily infiltrated by
Bar=25 fim. lymphocytes, sometimes aggregated in the lymphoid nodules
Figure 8. Nasal turbinate 6 d.p.i. (a) infiltrated lamina propria (arrow) and covered by an abnormal, flattened epithelium,
covered by a single thin epithelial layer, apparently still ciliated devoid of cilia. (H&E, bar = 25 pun).
(H&E). (b) TRTV antigen no longer detected. (PAP immunostain Figure 10. Nasal turbinate 21 d.p.i. Tissue recovery, but
consecutive section). Bar = 25 fxm. mucous glands still show a mild hypertrophy. (H&E, bar = 50
ion).

date seen in the lumen (Majö et al, 1995). The replication in the sinus tissue is substantial and
lesions we observed were always confined to the causes microscopical changes from 4 to 10 d.p.i.,
upper respiratory tract. Majö et al. (1995) also followed by deciliation, which lasts even longer.
found histological changes and detected TRT anti- These observations could support the work of
gen in the bronchi, but only in chickens inoculated Nakamura et al. (1997) who described the pathol-
via the trachea in addition to the eyedrop and ogy of swollen head syndrome in broiler chickens,
conjunctival routes. some of which were serologically positive for
The histopathological changes occurring in the TRT, whilst in others TRT antigen was detected
infra-orbital sinus following experimental avian using a reverse transcriptase polymerase chain re-
pneumovirus infection in chickens have not been action. They suggested that sinusitis appears to
reported previously. Our findings reveal that virus induce Escherichia coli proliferation in the subcu-
Sequential pathogenesis study of TRT in chickens 639

, • . i

• * - • , * *

Figure 11. Lower trachea (a) uninoculated 3-week-old chicken, H&E. (b) consecutive section, PAP immunostain, Light Green counter
stain, (c) lower trachea 5 d.p.i. showing thickening of the mucosa due to congestion, oedema and mononuclear cellular infiltration
in the lamina propria, associated with flattening of the epithelial cells, and focal deciliation (arrows) (H&E). (d) Five d.p.i. showing
one TRT-positive epithelial cell (arrow) (PAP immunostain, consecutive section). Bar = 25 urn.
Figure 12. Infra-orbital sinus mucosa, (a) 7 d.p.i. and (b) 5 d.p.i. Showing hyperplasia and focal exfoliation of the columnar ciliated
epithelium, accompanied by congestion, haemorrhages and mononuclear cell infiltration in the lamina propria (H&E). (c) 5 d.p. i TRTV
antigen in scattered columnar ciliated epithelial cells [PAP immunostain, consecutive section to (b)J. Bar = 25 ¡un.

taneous tissue as a result of destruction of respirat- Demonstration of antibodies to turkey rhinotracheitis virus in serum
from commercially reared flocks of chickens. Avian Pathology, 17,
ory epithelium and that TRT virus is a candidate
403^10.
for causing the initial damage. Cook, J.K.A., Holmes, H.C., Finney, P.M., Dolby, C.A., Ellis, M.M.
The results reported here provide the first & Huggins, M.B. (1989). A live attenuated turkey rhinotracheitis
detailed sequential examination of the dissemi- virus vaccine. 2. The use of the attenuated strain as an experimental
nation of a TRT virus in young chickens and vaccine. Avian Pathology, 18, 523-534.
describe the pathogenesis in that species. Previous Cook, J.K.A., Ellis, M.M. & Huggins, M.B. (1991). The pathogenesis
of turkey rhinotracheitis virus in turkey poults inoculated with the
studies in both turkeys and chickens have been virus alone or together with two strains of bacteria. Avian Pathol-
performed using an early UK TRT isolate of sub- ogy, 20, 155-166.
group A, made from turkeys. This, therefore, is Cook, J.K.A., Jones, B.V., Ellis, M.M., Jing Li. & Cavanagh, D.
also the first detailed study using a subgroup B (1993a). Antigenic differentiation of strains of turkey rhinotracheitis
isolate of TRT virus. virus using monoclonal antibodies. Avian Pathology, 22, 257-273.
Cook, J.K.A., Kinloch, S. & Ellis, M.M. (1993b). In vitro and in vivo
studies in chickens and turkeys on strains of turkey rhinotracheitis
virus isolated from the two species. Avian Pathology, 22, 157-170.
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isolation of a virus causing sinusitis in turkeys in South Africa and vaccine against different strains of turkey rhinotracheitis virus.
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Buys, S.B., du Preez, J.H. & Els, H.J. (1989). Swollen head syndrome Cook, J.K.A., Orthel, F., Orbell, S., Woods, M.A. & Huggins, M.B.
in chickens: a preliminary report on the isolation of a possible (1996). An experimental turkey rhinotracheitis (TRT) infection in
aetiological agent. Journal of the South African Veterinary Associ- breeding turkeys and the prevention of its clinical effects using
ation, 60, 221-222. live-attenuated and inactivated TRT vaccines. Avian Pathology, 25,
Cavanagh, D. & Barrett, T. (1988). Pneumovirus-like characteristics 231-243.
of the mRNA and proteins of turkey rhinotracheitis virus. Virus Jones, R.C., Baxter-Jones, C , Wilding, G.P. & Kelly, D.F. (1986).
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chicken trachéal organ cultures for the isolation and assay of avian 600.
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Cook, J.K.A., Dolby, C.A., Southee, D.J. & Mockett, A.P.A. (1988). G.P. (1987). Experimental infection of chickens with a ciliostatic
640 E. Catelli et al.

agent isolated from turkeys with rhinotracheitis. Veterinary Record, importantes et régressaient rapidement. Cette étude, basée sur
120, 301-302. l'utilisation d'un isolat pathogène de TRTV provenant de poulets
Jones, R.C., Williams, R.A., Baxter-Jones, C , Savage, C.E. & Wilding, malades, prouve clairement que le virus TRT peut entraîner des lésions
G.P. (1988). Experimental infection of laying turkeys with rhinotra- au niveau du tractus respiratoire du poulet et que ces lésions sont à la
cheitis virus: distribution of virus in the tissues and serological fois localisées et de courte durée.
response. Avian Pathology, 17, 841-850.
Jones, R.C., Naylor, C.J., Bradbury, J.M., Savage, C.E., Worthington,
K. & Williams, R.A. (1991). Isolation of a turkey rhinotracheitis-like ZUSAMMENFASSUNG
virus from broiler breeder chickens in England. Veterinary Record, Studie über die Ausbreitung eines Hühnerisolats des aviären
129, 509-510. Pneumovirus in Hühnern mit Hilfe von Virusisolierung, histo-
Jönsson, L.G.O. & Engström, B.E. (1986). Immunohistochemical pathologischer Untersuchung und Immunoperoxidase-Test
detection of infectious bursal disease and infectious bronchitis viral
antigens in fixed, paraffin-embedded chicken tissues. Avian Pathol- Ein Isolat der Untergruppe B des Puten-Rhinotracheitisvirus (TRTV)
ogy, 15, 385-393. oder aviaren Pneumovirus (APV) aus einer Herde kommerzieller
Juhasz, K. & Easton, A.J. (1994). Extensive sequence variation in the Zuchthühner mit schlechter Legeleistung, Mortalität und Swollen-
attachment (G) protein gene of avian pneumovirus: evidence for two Head-Syndrom verursachte sowohl bei jungen SPF-Hühnern als auch
distinct subgroups. Journal of General Virology, 75, 2873-2880. bei Küken mit hohen Titern maternaler TRT-Antikörper erhebliche
Majó, N., Allan, G.M., O'Loan, C.J., Pages, A. & Ramis, A.J. (1995). respiralorische Symptome.
A sequential histopathologic and immunocytochemical study of Dieses Isolat vermehrte sich zu hohen Titern im Respirationstrakt
chickens, turkey poults and broiler breeders experimentally infected experimentell infizierter SPF-Küken ungefähr 5 Tage lang nach der
with turkey rhinotracheitis virus. Avian Diseases, 39, 887-896. Infektion, wurde aber nach dieser Zeit nur gelegentlich reisoliert. Es
Nakamura, K., Mase, M., Tanimura, N., Yamaguchi, S., Nakazawa, M. wurde niemals aus anderen Geweben als denen des Respirationstrakts
& Yuasa, N. (1997). Swollen head syndrome in broiler chickens in Isoliert. Eme detaillierte, sequentielle histologische und Immunoperox-
Japan: its pathology, microbiology and biochemistry. Avian Pathol- idase-Untersuchung wurde durchgeführt. Dabei zeigte sich, daß TRT-
ogy, 26, 139-154. Virus zwar durchweg im Epithel der oberen Luftwege nachgewiesen
O'Loan, C.J. & Allan, G.M. (1990). The detection of turkey rhinotra- konnte, wenn auch nur für eine kurze Zeit nach der Infektion, aber die
cheitis virus antigen in formalin fixed, paraffin embedded tissue Schädigung, die es bewirkte, war minimal und die Erholung ertolgte
using a streptavidin-biotin-immunoperoxidase method. Avian Path- rasch. Diese Studie, bei der ein von erkrankten Hühnern gewonnenes
ology, 19, 401-407. TRT-Isolat benutzt wurde, liefert einen eindeutigen Beweis dafür, daß
Picault, J-P., Giraud, P., Drouin, P., Guittet, M., Bennejean, G., TRT-Virus eine Schadiguno des Respirationstrakts von Hühnern verur-
Lamande, J., Toquin, D. & Gueguen, C. (1987). Isolation of a sachen kann, und daß diese Schädigung sowohl lokalisiert als auch
TRTV-Iike virus from chickens with swollen-head syndrome. Veter- kurzlebig ist.
inary Record, 121, 135.
Shu, S., Yu, G. & Fan, L. (1988). The glucose oxidase-D AB -nickel
method in peroxidase histochemistry of the nervous system. Neuro- RESUMEN
science Letters, 85, 169-171. Utililazacion de técnicas de aislamiento vírico, histopatologia y
tecnicas de inmunoperoxidas para el estudio de la diseminación de
una cepa de pneumovirus en pollos
RÉSUMÉ
Se demostró que una cepa del subgrupo B del virus de la rinotraqueítis
Utilisation des techniques d'isolement viral, d'histopathologie et
(TRTV) o pneumovirus aviar (APV), aislada a partir de un lote de
d'immunoperoxidase pour étudier la dissémination d'un pneu-
reproductores comerciales que presentaba un cuadro de disminución de
movirus chez le poulet
la puesta, mortalidad y síndromo de cabeza hinchada, era capaz de
Un isolat du sous-groupe B du virus de la rhinotrachéite infectieuse de producir una sintomatología respiratoria grave, tanto en pollos SPF
la dinde (TRTV) ou du pneumovirus aviaire (APV), provenant d'un como en pollos con un elevado nivel de anticuerpos maternos anti TRT.
troupeau commercial de poules reproductrices, présentant une pro- Esta cepa presentó elevados títulos de replicación en el tracto
duction insuffisante en oeufs, de la mortalité et un syndrome du respiratorio de los pollos SPF infectados experimentalmente durante los
gonflement de la tête, s'est avéré provoquer des signes respiratoires cinco días posteriores a la inoculación, mientras que sólo se pudo aislar
importants chez de jeunes poulets SPF et chez des poussins présentant ocasionalmente después de este periodo. En ninguna ocasión se pudo
des titres élevés en anticorps TRT d'origine maternelle. aislar el virus a partir de otros tejidos u órganos. Se realizó un detallado
Cet isolat s'est multiplié à un titre élevé au niveau du tractus estudio secuencial mediante la aplicación de técnicas histológicas y de
respiratoire pendant approximativement cinq jours après inoculation immunoperoxidasa. Con estas técnicas se evidenció que mientras la
expérimentale de poulets SPF, par la suite, il n'a été réisolé presencia del virus del TRT se podía demostrar claramente en el epitelio
qu'occasionnellement. Il n'a jamais été réisolé à partir d'autres tissus de las vías respiratorias altas (aunque sólo durante un corto periodo de
que celui du tractus respiratoire. Une étude détaillée séquentielle basée tiempo después de la inoculación), la lesion causada era mínima y de
sur l'histologie et sur l'immunoperoxydase a été entreprise. Elle a reciperación rápida. En este estudio, utilizando una cepa patógena de
démontré que le virus TRT pouvait être mis en évidence au niveau de TRT obtenida a partir de animales enfermos, se demuestra claramente
l'épithélium des tissus du tractus respiratoire supérieur pendant un que el virus del TRT puede causar lesiones en el tracto respiratorio de
temps court après l'inoculation et que les lésions induites étaient peu los pollos y que esta lesion es muy localizada y de corta duración.

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