Bournival, Véronique

Chénier, Sonia

Desroches, Michel

Gagnon, Carl A.

Grenier St-Sauveur, Valérie

Koszegi, Marika

Provost, Chantale

English

Université de Montréal

CVJ / VOL 60 / DECEMBER 2019 1285
Cross-Canada Disease Report  
Rapport des maladies diagnostiquées  
au Canada
D uck atadenovirus A, also known as Duck adenovirus 1 (DAdV-1), is a member of the genus Atadenovirus (1). 
This virus is also responsible for the Egg Drop Syndrome (EDS) 
of laying hens (2). This syndrome is listed as an immediately 
notifiable disease in Canada, because it is considered as an 
exotic disease for which there are no control or eradication 
programs (3).
Although this virus has never been reported in commercial 
chicken production in Canada, DAdV-1 was identified in 
2009 in 2 Muscovy duck farms in Ontario (4). An adenovirus 
was also isolated in goslings with respiratory clinical signs in 
Saskatchewan in 1992, but the adenovirus species was unde-
termined (5,6). Antibodies to DAdV-1 have been detected in 
numerous wild waterfowl in North America, up to 42% in 
sampled ducks, suggesting it is widespread on the continent 
and that these birds act as reservoirs (7). Overall, DAdV-1 seems 
weakly pathogenic for ducks and geese because most infections 
are asymptomatic, and the rare reports of clinical signs always 
imply very young (4 to 20 d of age) ducklings and goslings. 
When sick, these birds show gasping, dyspnea, coughing, and/or 
sneezing and mortality is moderate (2% to 7%) (4–6). DAdV-1 
is substantially more pathogenic in chicken layers, in which it 
is associated with a severe drop in egg production that could 
last for 4 to 10 wk, and the laying of thin-shelled, soft-shelled, 
or shell-less eggs (2).
The first clinical case of DAdV-1 in a commercial duck farm 
in Québec is described herein, with whole genome sequencing 
of the virus.
Case description
In June 2019, a duck producer called his veterinarian for mor-
tality associated with possible cough in 3-week-old ducklings. 
This producer owned a multi-age broiler duck farm located in 
Québec, on one site which had several buildings. Sick animals 
were part of a group of 325 birds, housed on the 2nd floor of 
a building in which they were separated from another group of 
2-week-old birds by a 16-inch plain barrier (same ventilation). 
This building also housed 2 lots of 10- and 11-week-old duck-
lings on the first floor. Except for the group of 3-week-old duck-
lings, no other group experienced clinical signs. Interestingly, 
only females (n = 25) were sick in the affected group and all 
other groups were composed of males only. It was the first time 
that this producer had bought females from his regular hatchery, 
to satisfy one of his clients who wanted this product.
Five 3-week-old dead female ducklings were submitted to the 
Laboratoire de Santé Animale de St-Hyacinthe. Gross examina-
tion revealed white soft plugs in their trachea and/or bronchi, 
obstructing their lumen (Figure 1). The tracheal mucosa was 
moderately congested. On histopathology, the tracheal epi-
thelium was severely hyperplastic, with extensive loss of cilia 
and glands. The mucosa was moderately to severely infiltrated 
by heterophils, lymphocytes, and plasma cells. In the bronchi, 
similar lesions were seen but the epithelium was also often 
eroded or sloughed and, in some epithelial cells, a basophilic 
intranuclear inclusion was found (Figure 2). The plugs were 
Québec
First reported outbreak of Duck atadenovirus A tracheobronchitis  
in 3-week-old ducklings in Québec including whole genome sequence  
of the virus
Sonia Chénier, Michel Desroches, Chantale Provost, Véronique Bournival,  
Valérie Grenier St-Sauveur, Marika Koszegi, Carl A. Gagnon
Laboratoire de santé animale de St-Hyacinthe, Ministère de 
l’Agriculture, des Pêcheries et de l’Alimentation du Québec 
(MAPAQ), 3220 Sicotte, St-Hyacinthe, Québec J2S 7X9 
(Chénier); Services vétérinaires Michel Desroches, 260, 
rue Principale, St-Cléophas-de-Brandon, Québec J0K 2A0 
(Desroches); Molecular diagnostic laboratory (MDL) and Swine 
and Poultry Infectious Diseases Research Center (CRIPA), 
Faculté de médecine vétérinaire (FMV) Université de Montréal, 
3200 Sicotte, St-Hyacinthe, Québec J2S 2M2 (Provost, 
Bournival, St-Sauveur, Koszegi, Gagnon).
Address all correspondence to Dr. Sonia Chénier; e-mail: 
Sonia.Chenier@mapaq.gouv.qc.ca or Dr. Carl A. Gagnon; 
e-mail: carl.a.gagnon@umontreal.ca
Use of this article is limited to a single copy for personal study. 
Anyone interested in obtaining reprints should contact the 
CVMA office (hbroughton@cvma-acmv.org) for additional 
copies or permission to use this material elsewhere.
FOR PERSONAL USE ONLY
1286 CVJ / VOL 60 / DECEMBER 2019
R
A
P
P
O
R
T
 D
E
S
 M
A
L
A
D
IE
S
 D
IA
G
N
O
S
T
IQ
U
É
E
S
 
A
U
 C
A
N
A
D
A
composed of necrotic debris, sloughed epithelial cells, degen-
erated leucocytes, and a mixed population of bacteria. An 
heterophilic granuloma containing fungal hyphae (compatible 
with Aspergillus) was detected in an intrapulmonary bronchus 
of 1 bird. Samples of trachea and lungs were tested by reverse 
transcriptase-quantitative polymerase chain reaction (RT-qPCR) 
for Newcastle disease (APMV-1) and influenza A viruses and 
were negative. Routine bacteriology of tracheal swabs and liver 
yielded contaminants only.
As part of the differential diagnoses, the presence of DAdV-1 
was suspected and therefore, samples were submitted to the 
Molecular Diagnostic Laboratory (MDL) of the Faculté de 
médecine vétérinaire for analysis. No specific DAdV-1 PCR 
assay was available at MDL. Nonetheless, a pan avian adenovirus 
PCR assay, adapted from Raue et al (8), was available. Briefly, 
this PCR assay targets the hexon gene of avian adenoviruses 
and when positive, the expected PCR product length should be 
approximately 593 nucleotide base pairs. This pan avian adeno-
virus PCR assay was used on a pool of tracheal swabs, trachea, 
and lung. Interestingly, a PCR amplicon of the expected size was 
obtained, confirming the presence of an adenovirus. To identify 
the adenovirus species, the PCR product was sequenced using 
the Sanger method. Sequence analysis confirmed that adenovirus 
species was in fact DAdV-1 since the PCR product possesses a 
nucleotide identity of 99.63% compared to the DAdV-1 refer-
ence strain EDS76.
Considering that no entire viral genome sequence of DAdV-1 
has been previously reported in Canada, we decided to estab-
lish whether our virus was similar to what has been previously 
reported worldwide. Thus, an attempt was made to sequence 
the entire viral genome directly from the clinical samples 
using MiSeq Illumina technology (Illumina, Vancouver, British 
Columbia) to allow us to better characterize our DAdV-1 
strain, named FMV19-2234581. Briefly, the tissue was lysed 
for 10 min using glass beads. Then, the tissue lysate was centri-
fuged at low speed to remove large particles. The supernatant 
was placed on a sucrose cushion and then ultracentrifuged in an 
AirFuge microultracentrifuge (Beckman Coulter, Mississauga, 
Ontario) for 1 h to concentrate the virus. The pelleted virus 
was resuspended in phosphate-buffered saline (PBS) and the 
DNA was extracted using the Zymo RNA/DNA extraction 
kit (Cedarlane, Burlington, Ontario). The DNA was quanti-
fied using a Qubit fluorometer with the HS DNA kit, then a 
library was generated with a Nextera XT kit (Illumina). The 
library was sequenced using a MiSeq apparatus with a 600 v3 
cartridge. The complete genome was obtained using the de novo 
metagenomic application plus the extend contigs option of the 
CLC Genomic Workbench, Version 12.0.3 software (Qiagen, 
Toronto, Ontario). The annotations were transferred using 
the reference strain Duck adenovirus 1 isolate D11-JW-032 
(KJ452172) in Geneious Prime software, Version 2019.2.1 
(Biomatters, Newark, New Jersey, USA). The nucleotide homol-
ogy and PHYLM phylogenetic tree were made from a MAFFT 
alignment.
From the 9 674 970 total reads obtained, 0.025% (n = 2419) 
mapped to DAdV-1. A mean coverage of 16.07 (with a range of 
minimum and maximum values of 1 to 85) was obtained. The 
DAdV-1 FMV19-2234581 strain possesses a double-stranded 
DNA genome and was found to have a size of 33 222 nucleo-
tides. The FMV19-2234581 nucleotide sequence was submitted 
to GenBank (accession number MN310513). The FMV19-
2234581 whole viral genome nucleotide sequence clustered 
Figure 1. Trachea of an affected duckling. A white fibrin plug 
is completely obstructing the tracheal lumen.
Figure 2. Bronchus of an affected duckling. There is hyperplasia 
and squamous metaplasia of the epithelium, infiltration by 
heterophils, and degeneration/necrosis and sloughing of 
individual to small groups of epithelial cells (black arrow). 
The lumen of the bronchus is filled with mucus, degenerated 
leucocytes, and necrotic epithelial cells and debris. The 
degenerated/necrotic epithelial cells contain a basophilic 
intranuclear inclusion body (white arrow and insert).
200 mm
FOR PERSONAL USE ONLY
CVJ / VOL 60 / DECEMBER 2019 1287
C
R
O
S
S
-C
A
N
A
D
A
 D
IS
E
A
S
E
 R
E
P
O
R
T
with the other DAdV-1 reference strains as expected (Figure 3). 
Overall, the nucleotide identity between FMV19-2234581 and 
the other DAdV-1 reference strains varied between 98.23% and 
98.33%.
After this outbreak, no new cases were reported in this group 
of ducklings and even when the producer bought other females 
throughout the year, no other groups of ducklings experienced 
clinical signs.
Discussion
This is the first outbreak of DAdV-1 tracheitis reported in 
Québec and only the second outbreak reported in Canada. It 
is unknown if this infection is common or not in commercial 
Canadian duck production because, to our knowledge, no 
extensive serologic survey for the detection of DAdV-1 anti-
bodies has been carried out on these farms. In fact, the com-
mercial DAdV-1 (Egg Drop Syndrome) ELISA test, designed 
for use in chickens, does not work on duck sera (Jantina De 
Vylder, Biochek, personal communication, 2019). In Europe, 
a hemagglutination inhibition test has been developed for use 
in ducks intended for export, but this test is not available in 
Canada (Hervé Morin, Filavie, personal communication, 2019). 
However, even if the virus is introduced into a flock, it seems 
moderately to weakly pathogenic because only 1 group of duck-
lings was affected at the farm, even though younger ducklings 
were housed on the same floor, with common ventilation and 
only separated from the affected group by a barrier. Thus, it is 
possible that this virus is more widespread than initially thought 
in commercial duck farms in Québec but remains undetected. 
Another interesting fact about this case is that only females 
Figure 3. Nucleotide phylogenetic tree of the entire viral genome of different types of 
adenoviruses infecting various species of birds. Sequences of all the strains that are present in the 
tree were obtained from GenBank, their accession number is written following their name. The red 
arrow represents the complete viral genome of the first DAdV-1 Canadian strain to be sequenced 
(i.e., FMV19-2234581). The phylogenetic tree was created using nucleotide alignment PhyML Tree 
with substitution model HKY85 and 1000 bootstraps, using MAFFT alignment in Geneious Prime 
software version 2019.2.1. The bootstrap values are indicated in the tree. The scale bar represents 
the amount of nucleotide substitutions per site between viral strains.
FOR PERSONAL USE ONLY
1288 CVJ / VOL 60 / DECEMBER 2019
R
A
P
P
O
R
T
 D
E
S
 M
A
L
A
D
IE
S
 D
IA
G
N
O
S
T
IQ
U
É
E
S
 
A
U
 C
A
N
A
D
A
experienced clinical signs. This apparent sex-related sensitivity 
has never been reported in the literature. We could hypothesize 
that female ducklings responded differently to the virus when 
exposed to this agent for the first time. However, no females 
arriving at the farm after this episode became ill.
Interestingly, the FMV19-2234581 strain seems to be a 
more variable strain compared with other strains. In fact, the 
nucleotide identities of the other DAdV-1 strains varied between 
99.738% and 100% when compared with each other, which is 
greater than when those strains are compared with FMV19-
2234581 (98.234% to 98.327%). At present, it is difficult to 
establish if FMV19-2234581 could be a new virus subtype of 
DAdV-1 but further analyses will be required to determine this. 
All DAdV-1 strains included in the phylogenetic tree, how-
ever, originate from quite different geographical regions (i.e., 
China, Ireland, South Korea) compared with FMV19-2234581 
(Canada). This geographical distance may have contributed to 
the increase in genomic evolution and diversity of the FMV19-
2234581 strain compared with other DAdV-1 strains.
Acknowledgments
This work was funded by the Ministère de l’Agriculture, des 
Pêcheries et de l’Alimentation du Québec (MAPAQ) and 
Molecular Diagnostic Laboratory, Faculté de médecine vétéri-
naire, Université de Montréal. Dr. Carl A. Gagnon was finan-
cially supported by a Natural Sciences and Engineering Research 
Council of Canada (NSERC) discovery grant.
References
1. International Committee on Taxonomy of Viruses (ICTV). Spreadsheet of 
Current Taxonomy, Master species list, 2018v v2. Available from: https://
talk.ictvonline.org/files/master-species-lists/m/msl/8266 Last accessed 
October 25, 2019.
2. Smyth JA. Atadenovirus (egg drop syndrome and related infections). In: 
Swayne DE, ed. Diseases of Poultry. 13th ed. Hoboken, New Jersey: John 
Wiley and Sons, 2013:301–309.
3. Canadian Food Inspection Agency, Government of Canada. Immediately 
Notifiable Diseases, 2018. Available from: http://www.inspection.gc.ca/ 
animals/terrestrial-animals/diseases/immediately-notifiable/eng/1305670 
991321/1305671848331 Last accessed October 25, 2019.
4. Ivanics É, Palya V, Glávits R, et al. The role of egg drop syndrome virus 
in acute respiratory disease of goslings. Avian Pathol 2001;30:201–208.
5. Brash ML, Swinton JN, Weisz A, Ojkić D. Isolation and identification of 
duck adenovirus 1 in ducklings with proliferative tracheitis in Ontario. 
Avian Dis 2009;53:317–320.
6. Riddell C, den Hurk JV, Copeland S, Wobeser G. Viral tracheitis in 
goslings in Saskatchewan. Avian Dis 1992;36:158–163.
7. Gulka CM, Piela TH, Yates VJ, Bagshaw C. Evidence of exposure of 
waterfowl and other aquatic birds to the hemagglutinating duck adeno-
virus identical to EDS-76 virus. J Wildl Dis 1984;20:1–5.
8. Raue R, Gerlach H, Müller H. Phylogenetic analysis of the hexon loop 
1 region of an adenovirus from psittacine birds supports the existence of 
a new psittacine adenovirus (PsAdV). Arch Virol 2005;150:1933–1943.
PLATINUM SPONSOR/COMMANDITAIRE PLATINE
CVMA Corporate Partnership Program 
Programme de Partenariats de L’ACMV
The CVMA would like to extend recognition to the following sponsors for their overall 
contribution to the  Association for 2019.
L’ACMV tient à remercier les commanditaires suivants de leur contribution générale 
au travail de l’Association en 2019.
GOLD SPONSORS/COMMANDITAIRES OR
SILVER SPONSORS/COMMANDITAIRES ARGENT
FOR PERSONAL USE ONLY


First reported outbreak of Duck atadenovirus A tracheobronchitis in 3-week-old ducklings in Québec including whole genome sequence of the virus

Köszegi, Marika

Dépôt Institutionnel Numérique

https://papyrus.bib.umontreal.ca/xmlui/bitstream/1866/22714/1/Chenier%20et%20al%202019.pdf

First reported outbreak of Duck atadenovirus A tracheobronchitis in 3-week-old ducklings in Québec including whole genome sequence of the virus

Abstract

Similar works

Full text

Available Versions

Université de Montréal

Dépôt Institutionnel Numérique