Isolation of chicken astrovirus from specific pathogen-free chicken
embryonated eggs
Luis Fabian N. Nuñez,∗ Silvana H. Santander Parra,∗ Elena Mettifogo,∗ Márcia Helena B. Catroxo,†
Claudete S. Astolfi-Ferreira,∗ and Antonio J. Piantino Ferreira∗,1
∗
Department of Pathology, School of Veterinary Medicine, University of São Paulo, Av. Prof. Dr. Orlando M.
Paiva, 87, 05508-270, São Paulo, SP, Brazil; and † Laboratory of Electron Microscopy, Center for Research and
Development of Animal Health, InstitutoBiológico, Av. Cons. Rodrigues Alves, 1252, 04014-002, São Paulo, SP,
Brazil
were screened for CAstV, and differential diagnostic testing was performed for avian nephritis virus,
avian rotavirus, avian reovirus, chicken parvovirus, infectious bronchitis virus, and fowl adenovirus Group
I to detect co-infection with other infectious agents.
Seven- or 14-day-old CEEs presented with hemorrhages, edema, a gelatinous aspect, deformities, and
dwarfism. The supporting membranes did not show
any alterations. Here, we have described the isolation
of CAstV and its pathological characteristics in SPF
CEE.
ABSTRACT Astroviruses have been associated with
enteric disorders in many animal species, including
chickens. Here, we describe the isolation, propagation, and pathological characteristics of chicken astrovirus (CAstV) in specific pathogen free (SPF)
chicken embryonated eggs (CEE) from chickens with
diarrhea and runting–stunting syndrome. The CEE
were inoculated via the yolk sac route. Viral confirmation was carried out using PCR techniques and
transmission electron microscopy negative staining
with ammonium molybdate. The intestinal contents
Key words: chicken astrovirus, chicken embryonated eggs, viral isolation, enteric virus
2015 Poultry Science 94:947–954
http://dx.doi.org/10.3382/ps/pev086
INTRODUCTION
ogy (Maondez and Arias, 2007; Kang et al., 2012b). Its
genome varies from 6.4 to 7.7 kb in length and has 3
open reading frames (ORF) and a poly-A tail (Koci
and Schultz-Cherry, 2002; Maondez and Arias, 2007;
De Benedictis et al., 2011). ORF 1a and ORF 1b encode nonstructural proteins, and ORF 2 encodes a capsid precursor protein (De Benedictis et al., 2011). The
family Astroviridaeis divided into 2 genera: mammalian
astroviruses and avian astroviruses. CAstV has been associated with RSS in chickens and has been associated
previously with poor weight gain and enteric diseases in
chickens. RSS was first described by Olsen (1977) and
Kouwenhoven et al. (1978) in broilers. At that time,
the syndrome produced a high economic impact due to
weight loss, lower feed conversion, and mortality. The
disease is characterized by diarrhea, depression, and
lethargy, with pale intestines and/or cecum containing
watery contents (Jindal et al., 2009). Antigenic characterization of avian astroviruses has been limited due
to the difficulty of isolating and growing these viruses
(Koci and Schultz-Cherry, 2002). Currently, 2 serotype
of CAstV has been described (Pantin-Jackwood et al.,
2011).
Here, we report the isolation of CAstVs and their
pathological features in specific pathogen free (SPF)
chicken embryonated eggs (CEE) obtained from commercial chicken flocks with diarrhea and RSS.
Enteric poultry diseases are reported worldwide, such
as runting–stunting syndrome (RSS) and malabsorption syndrome in broilers and poult enteritis syndrome
and poult enteritis and mortality syndrome in turkey
poults. These enteric syndromes are associated with
several infectious agents, including viruses and bacteria, although viruses are being more frequently studied
and associated with enteric diseases in chickens (Saif,
2013; Kang et al., 2012a; Reynolds and Schultz-Cherry,
2008).
Astroviruses have been detected and associated with
enteric diseases (Reynolds and Schultz-Cherry, 2008).
Five avian astroviruses have been identified in poultry: turkey astroviruses 1 and 2 [by Saif et al. (1985)],
chicken astrovirus [CAstV; by Baxendale and Mebatsion (2004)], avian nephritis virus [ANV; by Imada
et al. (2000)], and duck astrovirus [by Asplin (1965)],
being their genomes completely sequenced.
CAstV is a small, rounded, nonenveloped virus, typically 28 to 30 nm in diameter with a star-like morphol
C 2015 Poultry Science Association Inc.
Received November 4, 2014.
Accepted January 15, 2015.
1
Corresponding author: [email protected]
947
948
NUÑEZ ET AL.
Table 1. Target genes, primer sequences, and amplicon sizes for the RT–PCR and PCR assays for differential diagnostic testing for
enteric viruses, including CAstV.
Virus
Primer names Gene target
ChPV
CAstV
ANV
FAdV-I
IBV
Avian reovirus
Avian rotavirus
NDV
PVF1
PVR1
CAS pol 1F
CAS pol 1R
ANV pol 1F
ANV pol 1R
Hexon A
Hexon B
UTR 11
UTR 44
UTR 31
S4-F13
S4-R1133
NSP4-F30
NSP4-R660
New S
New A
NS
ORF-1b
ORF-1b
Hexon
UTR
S4
NSP4
F
Primer sequences
Amplicon size (bp)
Reference
561
Zsak et al., 2009
362
Day et al., 2007
473
Day et al., 2007
897
Meulemans et al., 2001
179
Cavanagh et al., 2002
1,120
Pantin-Jackwood et al., 2008
630
Day et al., 2007
324
Pang et al., 2002
5 TTCTAATAACGATATCACT3
5 TTTGCGCTTGCGGTGAAGTCTGGCTCG3
5 GAYCARCGAATGCGRAGRTTG3
5 TCAGTGGAAGTGGGKARTCTAC3
5 GYTGGGCGCYTCYTTTGAYAC3
5 CRTTTGCCCKRTARTCTTTRT3
5 CAARTTCAGRCAGACGGT3
5 TAGTGATGMCGSGACATCAT3
5 ATGTCTATCGCCAGGGAAATGTC3
5 GGGCGTCCAAGTGCTGTACCC3
5 GCTCTAACTCTATACTAGCCTA3
5 GTGCGTGTTGGAGTTTCCCG3
5 TACGCCATCCTAGCTGGA3
5 GTGCGGAAAGATGGAGAAC3
5 GTTGGGGTACCAGGGATTAA3
5 GGAGGATGTTGGCAGCATT3
5 GTCAACATATACACCTCATC3
MATERIALS AND METHODS
Virus Strains
Intestinal contents of broilers or broiler breeders
showing signs of enteric disease were sent to the Laboratory of Avian Diseases at Faculdade de Medicina
Veterinária e Zootecnia–University of São Paulo for
molecular diagnosis of enteric viruses [CAstV, ANV,
reovirus, rotavirus, fowl adenovirus Group I (FAdV1), infectious bronchitis virus (IBV), and chicken parvovirus (ChPV)]; sequences of primers used are described in Table 1. The procedure is described by Mettifogo et al., 2014; 280 samples were collected between
2008 and 2010 from broilers or broiler breeders with
clinical signs of diarrhea or RSS from farms located in
several Brazilians states (São Paulo, Santa Catarina,
and Rio Grande do Sul), which are areas of intensive
poultry production. Each of these samples was tested
for all 7 viruses described above according to previously described methods (Mettifogo et al., 2014) and 14
were shown to be positive only for CAstV using realtime (RT)–PCR and confirmed by DNA sequencing.
These 14 samples positive only for CAstV were used in
the present study for isolation of CAstV with the inoculation of them in SPF CEE (Table 3).
CAstV Inocula
First-passage inocula were prepared using the same
suspension of the 14 samples positives only for CAstV
that was used for molecular testing, with the addition
of PBS 0.1 M at pH 7.4 to dilute the suspension to
1.5 mL, thus replacing the volume used for the DNA and
RNA (250 μL) extractions. The suspensions for molecular testing were made using an aliquot of intestinal
contents placed into sterile microfuge tubes containing
PBS 0.1 M at pH 7.4; these suspensions were homogenized and underwent 3 cycles of freezing and thawing
at −20◦ C for 10 min and 56◦ C for 1 min, respectively.
The samples were then centrifuged at 12,000 × g for
20 min at 4◦ C and the supernatant was diluted 1:7 to
a final volume of 1,300 μL with PBS 0.1 M at pH 7.4.
In addition, 10 μL antibiotic (10,000 IU/mL penicillin
and 2 mg/mL streptomycin sulfate) and 10 μL of antifungal (amphotericin B 0.02 mg/mL) were added to
each sample. For the next 2 viral isolation passages, a
suspension was made with equal aliquots of PBS and
macerated embryo and yolk collected during necropsy,
following the procedure described above.
SPF Chicken Embryonated Eggs
Seven-day-old SPF White Leghorn CEE were used.
Eggs were maintained in an automated hatchery that
provided both stable temperature (37.5 to 38◦ C) and
humidity (35 to 40%). The SPF CEEs were kindly doR
nated by CEVA
Animal Health and Laboratório Bio
Vet, both located in Brazil.
Virus Isolation
Seven-day-old (first group) and 14-day-old (second
group) CEEs were used for virus isolation. On the seventh or 14th d incubation, each egg was visualized, and
dead eggs were discarded. Viable eggs were marked with
the inoculation site, the sample number, and the passage number. The eggs were disinfected with 70% alcohol, drilled, and inoculated with 0.2 mL inocula using the yolk sac route. No other route of inoculation
was tested. A negative control was also inoculated with
0.2 mL sterile PBS 0.1 M at pH 7.4. Each sample was
inoculated into 3 eggs, and the eggs were sealed and
set in the hatchery. The viral samples were inoculated
949
ISOLATION OF CHICKEN ASTROVIRUS
Table 2. Results of CAstV isolation in SPF CEEs confirmed by RT–PCR and visualization of viral particles
by transmission electron microscopy, along with differential diagnostic testing for other enteric viruses.
Strategy for CAstV isolation in SPF CEE
Virus
Inoculation of 7-day-old CEEs
n = 14
Inoculation of 14-day-old CEEs
n = 14
Total number of viruses isolated
CAstV
ChPV
IBV
ANV
FAdV-1
Avian rotavirus
Avian reovirus
NDV
31
0
0
0
0
0
0
0
51
0
0
0
0
0
0
0
8
0
0
0
0
0
0
0
Total
3
5
8
1
Number of positive samples.
in 2 groups. In the first group (Group 1), inoculations
were performed in 7-day-old CEEs, when the formation
and differentiation of the embryonic intestines begin
(Senne, 1998); in the second group (Group 2), inoculations were performed in 14-day-old CEEs, when the
intestines are more developed and functional (Moran,
2007; Karcher and Applegate, 2008). Embryonic viability was observed daily for 5 d postinoculation.
Embryos that died within 24 h after inoculation were
discarded because they were considered to have a nonspecific cause of death, possibly due to damage caused
during inoculation or from bacterial contamination. After the first 24 h, dead eggs were taken out of the hatchery and maintained at 4◦ C until necropsy to avoid viral
degradation. In our experience, storing eggs in a refrigerator does not change the morphological characteristics of the embryo. Eggs inoculated on the seventh or
14th d age that maintained their viability until the fifth
d postinoculation were euthanized. For both eggs that
died during incubation and those that were euthanized,
necropsies were performed to examine the embryo and
yolk. The head, wings, and legs were removed before
the embryo was macerated and used for the next viral
passage. In parallel, after maceration, an aliquot of each
sample (embryo and yolk) was placed into a microfuge
tube containing 0.1 M PBS at pH 7.4, homogenized,
and submitted to 3 cycles of freezing and thawing. The
remaining procedures for making the inocula are described above in the preceding section; thereafter, a 0.2
mL volume of each sample was inoculated into eggs for
subsequent passages (Senne, 1998).
Macroscopic Characterization
SPF CEEs were subjected to necropsies after natural death or euthanasia. The embryos were examined for gross lesions that developed due to viral infection, such as deformities, stunting, and curled, as well
as the presence of hemorrhages, edema, and a gelatinous aspect. Macroscopic lesions were evaluated qualitatively to determine the presence (positive) or absence
(negative) of injuries.
Confirmation of Viral Isolation
Confirmation of viral isolation was carried out by detecting viral nucleic acid using the molecular techniques
of PCR and RT–PCR using primers and protocols cited
in Table 1, by visualizing viral particles using negative
staining electron microscopy (EM) and by determining
the presence of gross lesions in the embryo. Embryos
and supporting membranes that showed macroscopic
lesions that were positive by molecular techniques and
that had visible viral particles on EM were considered positive for CAstV isolation. At each passage, all
samples were also molecularly tested for ANV, ChPV,
IBV, FAdV-I, reovirus, rotavirus, and Newcastle disease
virus (NDV; Table 2).
Sampling, Preservation, and Processing
of Samples for Molecular Testing
RT–PCR and PCR were used to confirm RNA and
DNA viral isolation, respectively, in the CEEs containing macroscopic lesions and alterations in the embryonic tissues. The embryo and yolk were collected separately. After maceration, the samples were placed into
sterile 15 mL plastic tubes and stored at −20◦ C until
use. Also after maceration, an aliquot of each sample
(embryo and yolk) was collected in a microfuge tube
containing 0.1 M PBS at pH 7.4. Surplus material was
stored at −20◦ C. Samples collected in microfuge tubes
were frozen at −20◦ C for 10 min, thawed at 56◦ C for
1 min, and then homogenized for 20 s; this procedure
was repeated 3 times. The samples were then centrifuged at 12,000 × g for 20 min at 4◦ C for DNA viruses
or at 12,000 × g for 30 min at 4◦ C for RNA viruses.
Nucleic Acid Extraction
TRIzol reagent (Invitrogen Life Technologies, CA)
was used according to the manufacturer’s instructions
for the extraction of RNA and DNA from the samples and from negative (ultra-pure water) and positive
controls.
950
NUÑEZ ET AL.
Reverse Transcription Polymerase Chain
Reaction
The extracted RNA underwent reverse transcription
to obtain cDNA that was specific for a gene fragment
from each virus of interest. The reverse transcription reaction contained 3.5 μL extracted RNA and 6.5 μL reaction mix containing 2 μL 10× buffer, 1 μL 10 mM deoxynucleotide triphosphates, 1 μL dithiothreitol, 0.5 μL
the Moloney murine leukemia virus, enzyme (Invitrogen
Life Technology, CA) and 1 μL each of the forward and
reverse primers. The extracted RNA (3.5 μL) was denatured at 94◦ C for 5 min before addition to the reaction
mixture. The reverse transcription reaction was performed under the following conditions: 45◦ C for 60 min
and 72◦ C for 10 min. The cDNA then underwent PCR.
PCR
The PCR reaction used 22.5 to 24 μL mixture that
contained 0.5 pmol each of the forward and reverse
primers, 2.5 μL 10X buffer, 4 μL 1.25 mM deoxynucleotide triphosphates, 0.75 to 2 μL 50 mM MgCl2 ,
1 U platinum DNA polymerase (Invitrogen Life Technology, CA) and 1 to 2.5 μL cDNA. PCR was performed under the following conditions: 94◦ C for 3 min
to completely denature the cDNA; 30 cycles denaturation at 94◦ C for 1 min, hybridization of primers at 50◦ C
for 1 min, and extension at 72◦ C for 1 min; and a final
incubation at 72◦ C for 10 min. The reaction was then
maintained at 4◦ C for an unspecified period until being
stored at −20◦ C. The RT–PCR and PCR amplifications were performed using similar protocols to those
described previously, as listed in Table 1.
The amplified products (362 bp for CAstV, 473 bp
for ANV, 179 bp for IBV, 630 bp for avian rotavirus,
1,120 bp for avian reovirus, 561 bp for ChPV, 897
bp for FAdV-1, and 324 bp for NDV) were subjected
to electrophoresis in a 1.5% agarose gel along with a
100 bp molecular ladder (Invitrogen). The samples were
stained with Sybr Safe (Invitrogen) and analyzed with
a transilluminator and camera made by Alpha Imager
mini Analyses System (Alpha Innotech Corp., San Leandro, CA).
Negative Staining Transmission Electron
Microscopy
A suspension of each sample from both seven- and
14-day-old embryo homogenate preparations inoculated
with CAstV, as described above, was clarified at 1,780
× g for 10 min. The harvested supernatant was then
centrifuged at 100,000 × g for 90 min. The pellet was
resuspended in 0.5 mL deionized water, 3 μL was mixed
with 100 μL ammonium molybdate, and a 2 μL drop
of this mixture was placed onto a 200 mesh formvarcoated grid for 5 min. Grids were examined by an elec-
tron microscope (Philips, Model EM 208, Amsterdam,
Netherlands) to check for the presence of viral particles.
RESULTS
As is described in the materials and methods of
the original samples analyzed previously by own team
group (Mettifogo et al., 2014) only 14 were positives for
CAstV and negative for the other enteric viruses tested.
Virus Isolation
Virus isolation was confirmed by visualizing viral particles by EM, by the presence of gross lesions in the
embryo, and by using PCR and RT–PCR. All samples were tested for CAstV, ChPV, ANV, IBV, FAdVI, avian rotavirus, avian reovirus, and NDV. Here, we
isolated 8 samples of CAstV that were negative for isolation of ANV, ChPV, IBV, FAdV-I, NDV, rotavirus,
and reovirus. CAstV was isolated and propagated in 7and 14-day-old chicken embryonated eggs. Significant
lesions were observed after one passage in the 7-day-old
embryos, but no lesions were observed in the 14-day-old
embryos. Three CAstV samples were isolated and propagated in 7-day-old eggs, and 5 samples were isolated
from 14-day-old eggs (Tables 2 and 3). All samples positive by molecular techniques that were submitted for
negative staining EM showed viral particles, confirming
the presence of CAstV in the samples, as shown in Figure 2 (a and b represent strain 358-2 isolated in 7- and
14-day-old CEE, respectively). Negative controls were
negative for all viruses tested.
Strategy for Isolation, Culture, and
Propagation of Enteric Viruses in SPF CEE
For propagation in SPF CEE, samples potentially
containing enteric viruses were inoculated using the
yolk route, which is most commonly used for the propagation of enteric viruses. Viral strains of CAstV were
isolated in Groups 1 and 2 SPF CEE. There was better
isolation, culture, and propagation when the inoculum
was prepared as described in the Materials and Methods
section above. This strategy of isolation, culture, and
viral propagation was used in both groups, and CAstV
was isolated in the first passage. Testing suspensions for
multiple viruses using molecular techniques to identify
samples that are positive for only one virus is a strategy that can be used to isolate and propagate CAstV,
especially for the isolation of pure strains (Table 2).
Macroscopic Characterization of Lesions
Produced by CAstV in the CEE
The embryos were sacrificed according to the ethical protocols approved by the committee on Care and
Use of Laboratory Animal Resources of the School of
Veterinary Medicine, University of Sao Paulo and then
951
ISOLATION OF CHICKEN ASTROVIRUS
Table 3. Clinical history and demographic information for chicken flocks presenting with diarrhea and RSS signs for
which CAstV isolation was performed in SPF CEE.1
Individual clinical signs of
the birds from which the
samples come, and that were
inoculated in SPF CEE
CAstV isolated in SPF CEE
Sample
designations
Origin of the
sample
Chicken ages
Location, Brazilian
state
Diarrhea
Stunting
d7
d 14
242-62A
336-18
358-9E
354-6
359-1
359-2
359-3
358-22
361-1
361-2
362-1
362-5
371-3
388-1
Broilers
Broilers
Broilers
Broilers
Broiler breeders
Broiler breeders
Broiler breeders
Broilers
Broilers
Broilers
Broilers
Broilers
Broilers
Broilers
5 wk
8d
8d
29 d
1d
1d
1d
8d
8d
8d
3d
14 d
22 d
8d
SP
SP
SP
SC
SC
SC
SC
SC
SP
SP
RS
RS
RS
SC
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
Yes
No
No
No
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
NI
I
NI
NI
NI
I
NI
I
NI
NI
NI
NI
NI
NI
NI
I
I
NI
NI
I
NI
I
NI
NI
NI
I
NI
NI
1
I = Samples where CAstV was isolated in SPF CEE; NI = Not isolated from 7-day-old or 14-day-old CEEs; P = São Paulo State;
SC = Santa Catarina; RS = Rio Grande do Sul.
2
CAstV strain 358-2 was subjected to transmission electron microscopy.
Figure 1. A normal embryo (left) compared with hemorrhagic, edematous embryos with gelatinous aspects and dwarfism (right).
examined for lesions and macroscopic alterations. The
embryos from Group 1 showed lesions and macroscopic
alterations, including hemorrhages, edema, dwarfism,
a gelatinous aspect, and deformities. Macroscopic lesions were evaluated qualitatively as the presence (positive) or absence (negative) of lesions. The embryos from
Group 2 did not show any type of lesion or alteration.
Figure 1 shows embryonic lesions from CEEs where
CAstV was isolated, and Tables 2 and 3 provide viral
isolation strategies, results of the differential diagnostic
testing for other enteric viruses, clinical signs, and embryo ages. In 3 passages, there was no mortality at any
day postinoculation. The negative control embryos did
not show any lesion or macroscopic alteration.
Standards for the Macroscopic
Characterization of CAstV
In the 3 first passages, the embryos were completely
hemorrhagic and edematous with deformities, and to
952
NUÑEZ ET AL.
Figure 2. Transmission electron micrographs of CAstV particles in embryo homogenate preparations. Figures 2a and 2b show CAstV Strain
358-2 isolated from 7- and 14-day-old CEE, respectively. The bar = 280 nm.
a lesser extent, a gelatinous aspect. Dwarfism was evident, but curl was not observed. These results are depicted in Figure 1. There was no embryo mortality post
inoculation in any passage where the virus was isolated.
No lesions were found in the embryos inoculated at
14-days-old, which appeared normal, including normal
thoracic and abdominal internal organs. There was no
mortality.
DISCUSSION
Enteric diseases in poultry generate considerate economic losses, and many enteric viruses are associated
with them. Although enteric viruses in poultry have
been recognized for decades, there is still a lack of
information about them. Viruses have been identified
in chickens and turkeys that have enteric diseases in
many countries, thus demonstrating their wide distribution (Pantin-Jackwood et al., 2007; Pantin-Jackwood
et al., 2008; de Wit et al., 2011; Kang et al., 2012a;
Koo et al., 2013). Astroviruses have been identified in
turkeys (Silva et al., 2008; Moura-Alvarez et al., 2013)
and chickens (Mettifogo et al., 2014) in Brazil, suggesting a possible relationship between astroviruses and enteric diseases in birds. Here, we described the isolation
of CAstV from chickens with enteric problems in Brazil.
Embryonic age is considered a determinant in
the success or failure of viral isolation. Therefore,
953
ISOLATION OF CHICKEN ASTROVIRUS
7-day-old embryonated eggs were selected for inoculation as the optimal age for viral isolation using the
yolk sac route (Senne, 1998). However, based on the
enteric nature of CAstV and the stage of embryo development, yolk sac inoculation was also carried out in
14-day-old embryonated eggs, which may provide better isolation because the intestines would already be
developed and functional (Moran, 2007; Karcher and
Applegate, 2008).
CAstV grew well in both seven- and 14-day-old CEE.
The 7-day-old CEE were already described as an optimal age for isolation of enteric viruses (Senne, 1998).
However, the inoculation of 14-day-old CEE may also
be an optimal age for isolation of CAstV because at this
age, the intestines of the embryo are more developed,
and the intestinal cells likely possess more receptors for
CAstV, thus enabling its replication and propagation
without producing injuries in others tissues or organs
of the embryo.
Isolation of CAstV from fecal samples is very difficult
because of the presence of other viruses, such as adenovirus, reovirus, or rotavirus, which also grow well in
CEE (Guy, 2008). Here, we isolated CAstV from fecal
samples, confirming that CAstV is circulating in Brazilian chicken flocks and maybe in Latin America poultry;
however, there are no reports of CAstV in other countries on this part of the continent (Nuñez and Ferreira,
2013).
The samples of CAstV isolated here were inoculated
by the yolk sac route, demonstrating that this route is
effective for CAstV isolation. The use of the yolk sac
as a route of infection and the enterotropic nature of
CAstV may allow CAstV to directly enter the digestive
organs of the embryo, particularly the intestine; thus,
this procedure provides an optimal medium for CAstV
culture, development, and propagation. The yolk has
the function of nourishing the embryo, and thus is in
direct contact with the embryo from a few h after fecundation (Nakazawa et al., 2006).
There was no mortality in any of the 3 passages in
either group, demonstrating that CAstV may not produce embryonic lethality, similar to what has previously
been described for turkey astrovirus (Tang et al., 2006).
Samples of CAstV were isolated in the first passage and remained in subsequent passages; CAstV isolation did not require blind passages, unlike for other
viruses, such as IBV and reovirus, that require one
or more blind passages before isolation (Guy, 2008;
Bányai et al., 2011; Saif, 2013). The embryos from
which CAstV was isolated showed hemorrhages, edema,
and dwarfism, similar to the alterations previously
described for turkey astrovirus (Reynolds and SchultzCherry, 2008). These macroscopic characteristics indicated that CAstV reached the embryo and caused
injuries in the embryo at 7 d age; however, the supporting membranes did not show any alterations.
In addition to observing gross lesions in the embryo
and supporting structures, viral isolation may be confirmed by molecular techniques. Here, viral detection
was performed using molecular tests for specific gene
sequences that have been previously described (Meulemans et al., 2001; Cavanagh et al., 2002; Day et al.,
2007; Pantin-Jackwood et al., 2008; Zsak et al., 2009)
and using negative staining transmission EM to visualize viral particles. PCR and RT–PCR was used to
detect and confirm the isolation of CAstV and to exclude the presence of other viral agents such as ANV,
reovirus, rotavirus, FAdV-I, IBV, and ChPV.
There is little information about CAstV isolation
from CEE, although the virus has been detected in commercial chicken flocks (Pantin-Jackwood et al., 2006,
2007; Guy, 1998; de Wit et al., 2011; Mettifogo et al.,
2014). The results obtained here show the lesions that
CAstV produces in embryos, which may contribute to
our understanding of CAstV in the future and its relationship with the pathogenesis of enteric diseases. Here,
the isolation of CAstV was possible. This successful isolation was obtained by using suspensions for molecular
testing to identify samples that were positive only for
CAstV. In at least 3 samples no virus was detected in
any of the passages, which may be due to a low viral
quantity in the samples, storage conditions of the samples, the total time of sample storage, difficulty in viral
adaptation to the CEE, and/or problems in detecting
other viruses (Senne, 1998).
In our study, the CEE age (7- or 14-d age) chosen
for inoculation provides an alternative method for the
isolation and propagation of CAstV. Macroscopic characterization of the lesions caused by CAstV will help in
its diagnosis and viral detection. While the embryonic
lesions for other viruses are very similar and none provide a pathognomonic sign for the detection and confirmation of CAstV isolation, the presence of lesions, such
as hemorrhages, edema, dwarfism, a gelatinous aspect,
and deformities, should be suggestive of CAstV. To our
knowledge, this study represents the first isolation of
CAstV in SPF CEE from chickens with enteric disease
in Brazil.
ACKNOWLEDGMENTS
The authors would like to thank the Fundação de
Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico for financial support of this study. A. J. Piantino Ferreira is the recipient of a Conselho Nacional
de Desenvolvimento Cientı́fico e Tecnológico fellowship.
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