viruses
Article
First Seroprevalence Survey of Avian Reovirus in Broiler
Breeders Chicken Flocks in Morocco
Ahmed Achhal Elkadmiri 1,2 , Amal Zhari 1 , Noura Aitlaydi 2 , Mohammed Bouslikhane 1 , Asma Fagrach 1 ,
Mohamed Mouahid 3 and Siham Fellahi 1, *
1
2
3
*
Citation: Achhal Elkadmiri, A.;
Zhari, A.; Aitlaydi, N.; Bouslikhane,
M.; Fagrach, A.; Mouahid, M.; Fellahi,
S. First Seroprevalence Survey of
Department of Pathology and Veterinary Public Health, Agronomy and Veterinary Institute Hassan II,
Rabat 10000, Morocco; achhal.cvrc@gmail.com (A.A.E.); amalzhari1999@gmail.com (A.Z.);
bouslikhanemed@yahoo.fr (M.B.); fagrachasma@gmail.com (A.F.)
Cabinet Tit Mellil, Tit Mellil 29640, Morocco; aitlaydinoura@gmail.com
Cabinet Mouahid, Temara 12000, Morocco; mohamedmouahid@gmail.com
Correspondence: fellahisiham2015@gmail.com or s.fellahi@iav.ac.ma
Abstract: Avian reovirus (ARV) is a prevalent infectious agent that has the potential to cause respiratory and gastrointestinal illnesses in poultry, leading to substantial financial losses in the poultry
sector. Until now, there have been no investigations conducted to examine the epidemiological status
of ARV infections in Morocco. The aim of this study was to investigate the seroprevalence of ARV
infections with respect to area, types of chickens (broiler breeder, and broiler), vaccination status, and
age of chickens. A total of 826 serum samples were collected from 36 broiler and broiler breeder flocks,
with 14 of them being unvaccinated, fromsix different regions of Morocco, namely Casablanca-Settat,
Rabat-Salé-Kénitra, Tanger-Tétouan-Al Hoceïma, Oriental, Marrakech-Safi, and Fez-Meknès between
2021 and 2022.These serum samples were screened using a commercial indirect ELISA ARV antibody
test kit (IDEXX REO). The study found that all tested flocks were positive for ARV-specific antibodies,
indicating that the virus was present in these flocks. Out of the 826 serum samples tested, 782 were
positive for ARV-specific antibodies. The overall prevalence of ARV infections in breeder and broiler
flocks was calculated to be 94.6% ± 0.78. To summarize, the current study provides evidence of the
widespread distribution of ARV infections in Morocco, suggesting that the poultry industry in the
country is highly infected with ARV.
Keywords: avian reovirus; seroprevalence; broilers; breeders; Morocco; ELISA
Avian Reovirus in Broiler Breeders
Chicken Flocks in Morocco. Viruses
2023, 15, 1318. https://doi.org/
10.3390/v15061318
Academic Editors: Yantao Wu,
Tao Yun and Chengcheng Zhang
Received: 11 May 2023
Revised: 30 May 2023
Accepted: 31 May 2023
Published: 2 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1. Introduction
Avian reovirus is a virus that belongs to the Reoviridae family and the Orthoreovirus
genus. It can infect various species of birds, including chickens, turkeys, ducks, and quails.
The virus can cause several diseases in birds, including viral arthritis/tenosynovitis, respiratory tract infections, and malabsorption syndrome [1–3]. Avian reovirus is transmitted from
bird to bird through contact with infected feces, respiratory secretions, or contaminated
surfaces [1,4]. The virus can also be spread vertically from infected hens to their offspring
through the egg [5]. The diagnosis of avian reovirus infection typically involves a combination
of clinical signs, gross and histopathological lesions, and laboratory tests, including virus
isolation, serology, and molecular methods [1,6]. It is important to use a combination of these
techniques for an accurate diagnosis of avian reovirus infection [6–9]. The early detection and
diagnosis of the virus can help in implementing appropriate control measures, including
vaccination and biosecurity practices, to prevent the spread of the virus and minimize its
impact on poultry production. There are currently no specific treatments for avian reovirus
infections, and prevention mainly relies on good management practices, including maintaining proper biosecurity measures, vaccination, and maintaining optimal environmental
conditions for birds. Vaccination is an effective method of controlling avian reovirus infec-
4.0/).
Viruses 2023, 15, 1318. https://doi.org/10.3390/v15061318
https://www.mdpi.com/journal/viruses
Viruses 2023, 15, 1318
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tions in poultry. There are several types of vaccines available for avian reovirus, including
live attenuated vaccines, inactivated vaccines, and subunit vaccines [10].
The seroprevalence of avian reovirus varies depending on several factors, including
the age and breed of the birds, management practices, and geographical location. In
general, seroprevalence is higher in older birds than in younger birds, as older birds have
had more opportunities for exposure to the virus [7,8]. Seroprevalence studies are usually
conducted using enzyme-linked immunosorbent assay (ELISA) or serum neutralization
tests to detect antibodies against avian reovirus in bird blood samples [9,10]. The results
of these tests can provide important information about the prevalence of the virus in
a population and help to guide disease control strategies. It is important to note that
seroprevalence does not necessarily indicate the presence or absence of disease, as birds
can have antibodies against the virus without showing any clinical signs. Therefore, a
combination of seroprevalence data and clinical observations is needed to fully assess the
impact of avian reovirus infections on a bird population [10].
In Morocco, avian reovirus presents a significant concern for the poultry industry,
and ongoing research is necessary to develop new control strategies and to improve our
understanding of the virus and its impact on bird health and production. This study, for
the first time, investigates the seroprevalence of Reovirus in broiler breeder and broiler
chickens in different regions in Morocco.
2. Materials and Methods
2.1. Sampling Protocol
In this study, a total of 826 sera were collected from 36 different broiler breeder and
broiler flocks in 6 different regions of Morocco (Casablanca-Settat, Oriental, Tanger-TétouanAl Hoceïma, Rabat-Salé-Kénitra, Marrakech-Safi, Fès-Meknès). In total, 379 serums were
collected in 2021 and 447 in 2022, according to the distribution of collection dates. Several
parameters, such as age, type of bird (broiler breeder, broiler), and vaccination status were
considered during the sample collection (Table 1). The age group distribution of the birds
from whom the 826 serums were collected was as follows: 1 day old for broilers and 1 day
old, 8 to 12 weeks, and 21 to 26 weeks for broiler breeders’ flocks.
The stratified random sampling strategy used in this study provided statistical representativity with a confidence level of 95% and a diagnostic test sensitivity of 86%. For the
6 Moroccan regions included in the study, the criteria “total poultry farms for each region”
helped to divide the flocks into 3 subgroups called strata, each of which represented a
density (high, medium, and low density). For a given theoretical seropositivity rate of 50%,
and according to the size of flocks in each region (approximately 10,000), the minimum
required sample size is determined to be 6 per flock for each age group but can be increased to
12 to maximize the chances of having reliable results. For each bird, a sample random selection
is applied to ensure an equal chance of being selected. Nevertheless, in some circumstances,
the sample size was reduced due to potential losses during collection or storage.
Table 1. Serological detection of ARV specific antibodies according to all parameters.
Sera
+
Sp (%) *
Mat ± sd
High
226
200
88.5%
2879.42 ± 154.1 c
Medium
454
447
98.5%
6135.27 ± 200 a
Low
146
135
92.5%
5000.08 ± 317.7 b
Vaccinated
519
482
Unvaccinated
307
300
Parameters
Density
Criteria
92.8%
Vaccination
status
[91–95] b
98%
[96–99] a
5340.60 ± 168
4542.05 ± 241
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Table 1. Cont.
Parameters
Criteria
Sera
+
Sp (%) *
94.52%
Breeders
712
673
Broilers
114
109
Breeders 1 day
172
170
Breeders 8–12 weeks
244
210
Breeders 21–26 weeks
296
293
Broilers 1 day
114
109
2021
379
371
2022
447
411
Casablanca-Settat
226
200
Fez-Meknès
107
104
Marrakech-Safi
269
265
Oriental
98
89
Rabat-Salé-Kénitra
78
78
Tanger-Tétouan-Al Hoceïma
48
46
East
98
89
North
155
150
Type of birds
[92.85–96.2]
95.61% ns
[91.8–99.43]
98.84% a
Age groups
[97.22–100]
86.07% b
[81.69–90.44]
98.99% a
[97.84–100]
95.61% a
[91.8–99.43]
97.89% a
Period of
collection
[96.44–99.34]
91.95% b
[89.41–94.48]
88.50% c
[84.3–92.69]
97.20% ab
[94.02–100]
98.51% a
Regions
[97.06–99.97]
90.82% bc
[85–96.64]
100% a
95.83% ab
[85–96.64]
90.82% b
[85–96.64]
96.77% a
Geographic location
[93.96–99.59]
98.51% a
South
269
265
West
304
278
Mat ± sd
ns
[97.06–99.97]
91.45% b
[88.29–94.61]
4975.55 ± 151.4 ns
5470.03 ± 345.8 ns
5180.78 ± 257.4 a
3766.13 ± 219.1 b
5853.26 ± 266.9 a
5470.03 ± 345.8 b
3956.05 ± 161.8 b
5966.07 ± 207.6 a
5136.41 ± 240.2 a
3838.80 ± 322.9 b
4977.81 ± 265 a
5385.33 ± 432.4 a
5590.12 ± 455.8 a
6078.62 ± 240.2 a
5385.34 ± 432.4
4532.43 ± 294.2
4977.81 ± 265
5252.83 ± 213.4
Sera: Total number of sera samples analyzed; +: positive sera; SP (%): seroprevalence percentage; MAT ± SD:
Mean antibody titer ± standard deviation; * Seroprevalence values are shown along with 95% confidence intervals.
Different superscripts letters (a , b , c ) in the same column indicate a significant difference (p < 0.05) between criteria
of each parameter; ns : non-significant.
2.2. Collection of Blood, Transportation, and Serum Preparation
Blood samples were obtained from the wing vein, using a 3 mL syringe, of the randomly selected apparently healthy birds. The blood was transferred into sterile tubes and
Viruses 2023, 15, 1318
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kept at room temperature on a slanted surface, which allowed the blood to clot, and the
resultant supernatant designed serum was harvested by centrifugation at 12,000× g for
5 min and then transferred into a clean Eppendorf tube. Each sample was labeled with an
appropriate alphanumeric code. The collected sera were stored at −20 ◦ C in preparation
for performing the ELISA.
2.3. Serology
In order to detect the ARV antibodies, the collected and processed sera samples
were analyzed using a commercially available ARV-specific indirect ELISA kit (IDEXX
Laboratories, Westbrook, ME, USA) containing ARV antigen-coated plates, in accordance
with the instructions supplied by the manufacturer.
In brief, 100 µL of serially pre-diluted test samples (1:500) was dispensed into the
96-well microtiter plates coated with ARV antigens, and previously individually filled
with the positive (PC) and negative controls (NC). The microplates were subsequently
incubated for 30 min in order to form an antibody–antigen complex. After removing the
solution content (unbound materials) from the wells, the microplates were washed 5 times
with 350 µL of distilled water, then 100 µL of horseradish peroxidase conjugate (GOAT
anti-chicken) was dispensed into each well, which binds to any attached chicken antibody,
and incubated for 30 min and then washed as previously described. Thereafter, 100 µL
of TMB (3,3’,5,5’-Tetramethylbenzidine) substrate was incubated in each well for 15 min,
which allowed the color to develop at room temperature. Finally, the reaction in the wells
was terminated by dispensing 100 µL of a blocking solution. An ELISA microtiter plate
photometer reader, equipped with a 650 nm filter, was used to measure, and record the
absorbance values and the optical density of each well.
2.4. Statistical Analysis
In order to investigate the seroprevalence of birds against various diseases, several
parameters were considered including age, type of birds, regions and their geographic
location, vaccination status, period of collection, and flock density. The seropositivity
of indirect ELISA antibody titers, as well as their means and standard deviations, were
calculated using a one-way analysis of variance (ANOVA) model. Additionally, ANOVA
and post hoc t-tests were used to determine whether there were any significant differences
in antibody titers within and between the different parameters investigated. Differences
were considered statistically significant when their probability (P) values were equal to or
less than 0.05. By utilizing these statistical techniques, it was possible to identify the factors
that influence seroprevalence, and to determine the effectiveness of vaccination programs
against avian reovirus in different regions and age groups of birds.
2.5. Interpretation of ELISA Results
For each serum tested, the following formula (OD value of sample—OD of NC)/(OD of
PC—OD of NC) was used to calculate the simple to positive S/P ratio, and by converting the
resultant S/P ratio, this equation Log10 Titer = 1.09 (log10 S/P) + 3.36 helped to measure the
endpoint titers. Samples with titers greater than 396 (S/P ratio > 0.2) are considered positive.
3. Results
3.1. Serological Detection of ARV Specific Antibodies
The study evaluated a total of 826 serum specimens collected from different regions
of Morocco to determine the prevalence of avian reovirus-specific antibodies. The results
showed that 782/826 of the specimens were positive for ARV-specific antibodies, indicating
a high prevalence of the virus in the sampled regions.
3.2. Geographical Location Effect
A further analysis of the prevalence rates in different regions revealed that all regions
had confirmed ARV-specific antibodies, with the highest rates being found in the Marrakech-
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Safi 265 (98.51%) and Rabat-Salé-Kénitra 78 (100%) regions. The prevalence rates were lower
in the Casablanca-Settat and Oriental regions, with rates of 88.5% and 90.82%, respectively.
Interestingly, when considering Tanger-Tétouan-Al Hoceïma and Fez-Meknès in the north,
Casablanca-Settat and Rabat-Salé-Kénitra in the west, Marrakech-Safi in the south, and
Oriental in the east, the prevalence rates were significantly higher in the north and south
regions of Morocco, with rates of 96.77% and 98.51%, respectively, compared to the east
(90.82%) and west regions (91.45%).
3.3. ARV Seroconversion and Vaccination Effect
The study also evaluated the seropositivity rates in vaccinated and unvaccinated
flocks, and found that the unvaccinated flocks had a higher seropositivity rate of 98%
compared to 92.8% in vaccinated flocks. Furthermore, the study compared the prevalence
of ARV-specific antibodies between breeding and broiler chickens and found no significant
difference (p-value = 0.63) between the two groups. Moreover, the results showed that
the highest antibody titer means were recorded in the vaccinated flocks, particularly in
Tanger-Tétouan-Al Hoceïma, with an average of 6078.62 ± 240.2. On the other hand, the
unvaccinated flocks from Fez-Meknès had the lowest antibody titer mean with a rate of
3838.8 ± 322.9. Interestingly, when the geographic location of these regions was taken
into account, the average antibody titer means of 5037.10 ± 301.25 did not exhibit any
significant differences.
3.4. Chronology Effect
Furthermore, the study evaluated the prevalence rates of ARV-specific antibodies over
sample collection periods and found that the prevalence was significantly higher in 2021
than in 2022. Among the 782 serum specimens that tested positive for avian reovirus, there
was a wide range of antibody titers, ranging from 403 to 22,372, with an average of 5320.84.
Moreover, the study found that the level of ARV antibody was highly variable among
individuals, as evidenced by 680 out of the 782 seropositive serum samples having titers
between 396 and 9999. Subsequently, the period of collection had a significant effect on the
antibody titers, with a higher mean titer in 2022 (5966.07) compared to 2021 (3956.05).
3.5. Density and Age Effects
The findings indicate that flock density has a significant impact on antibody titers,
with the medium density flocks showing the highest mean titer of 6135.27 ± 200, followed
by low-density flocks with a mean titer of 5000.08 ± 317.72, and high-density flocks with a
mean titer of 2879.42 ± 154.1. The difference between the mean titers of medium-density
and low-density flocks is statistically significant (p < 0.05), while the difference between
medium-density and high-density flocks is not significant.
The type of production, either breeders or broilers, did not demonstrate a significant effect (p-value = 0.22) on the antibody titers, with means of 4975.55 ± 151.4 and
5470.03 ± 345.8, respectively. Regarding the age of chickens, the results indicate that the
mean antibody titer varies significantly depending on the age of the chicken. Chickens aged
between 21 and 26 weeks had the highest mean titer of 5853.26 ± 266.9, followed by chickens aged 1 day (5325.41 ± 301.6), and those aged between 8and12 weeks (3766.13 ± 219.1).
3.6. Statistical Analyses
Table 1 provides a summary of the statistical analysis results on various parameters
that may affect antibody titers in chickens. The correlation coefficients between different
parameters and the test results (positive or negative) for this certain study are shown in
Table 2. Among the criteria, the vaccination status of the birds had a statistically significant
correlation (p < 0.01) with the test results, with a Pearson correlation coefficient of 0.104
(Figure 1). Another significant factor was the geographic region, with a negative correlation
coefficient of −0.146 (p < 0.001). Interestingly, there was a weak positive correlation
(p < 0.05) between the age of the birds and the test results, with a Pearson coefficient of
Viruses 2023, 15, 1318
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Viruses 2023, 15, x FOR PEER REVIEW
6 of 11
0.017. On the other hand, there was no significant correlation between the density of the
flock, the type of production, or the period of collection and the test results (Figure 1).
Table 2. Correlation coefficient (r) with different parameters.
Table 2. Correlation coefficient (r) with different parameters.
Vaccination
DensityVaccination
Age
Status
Age
Density
Regions
Regions
Status
Period of
Type of
Type of
Birds
Period
of
Collection
Collection
Birds
Pearson
Results
Results
Pearson
correlation −0.010−0.010
** −0.146
−0.146
0.114
correlation
NS NS 0.104 0.104
**
** ** 0.114
** **
coefficient
coefficient
Sig. (bilateral)
Sig. (bilateral)
N
N
0.769 0.769
826
826
−−0.132**
0.132 **
0.017
0.017NS
NS
0.003 0.003
0.0000.000
0.001
0.001
0.000
0.000
0.630
0.630
826 826
826 826
826
826
826
826
826
826
Sig.: significance; NS: non-significant; **: highly significantly different at p < 0.01.
Sig.: significance; NS: non-significant; **: highly significantly different at p < 0.01.
6
8-12 weeks
5
21-26 weeks
Broilers
98.99
86.07
1 day
97.22
95.61
Breeders
94.52
4
88.50
98.50
Low
92.50
Tanger-Tétouan-Al Hoceïma
95.83
3
Rabat-Salé-Kénitra
100
Oriental
90.82
Fez-Meknès
97.20
Marrakech-Safi
98.51
2
Casablanca-Settat
88.50
Unvaccinated
98.00
Vaccinated
1
PARAMETERS
High
Medium
92.80
2022
91.95
2021
97.89
80
90
100
SEROPOSITIVITY (%)
Figure 1. Seropositivity percentage according to investigated parameters.
Figure 1. Seropositivity percentage according to investigated parameters.
Discussion
4.4.Discussion
Avian reovirus
reovirus is
is aa viral
viral pathogen
in in
chickens
Avian
pathogen that
thathas
hasbeen
beenfrequently
frequentlydetected
detected
chickens
worldwide, causing a range of clinical symptoms, including stunted growth, enteritis, resworldwide, causing a range of clinical symptoms, including stunted growth, enteritis,
piratory symptoms, pericarditis, and myocarditis. ARV infections in poultry can lead to
respiratory symptoms, pericarditis, and myocarditis. ARV infections in poultry can lead to
significant economic losses in the poultry industry, resulting in reduced productivity and
significant economic losses in the poultry industry, resulting in reduced productivity and
increased mortality. Despite the global prevalence of ARV infection in poultry, there is a
increased mortality. Despite the global prevalence of ARV infection in poultry, there is a
lack of information on its prevalence and impact on poultry farms in Morocco.
lack of information on its prevalence and impact on poultry farms in Morocco.
The proposed study is of great importance as it will be the first to investigate the
The proposed study is of great importance as it will be the first to investigate the
prevalence, transmission, and impact of ARV infections on poultry production in Moprevalence, transmission, and impact of ARV infections on poultry production in Morocco.
rocco. The study involved collecting and analyzing samples from poultry farms across
The
studyregions
involved
collectingtoand
analyzing
samples from
poultry
farmsAdditionally,
across different
different
of Morocco
determine
the prevalence
of ARV
infections.
regions
of
Morocco
to
determine
the
prevalence
of
ARV
infections.
Additionally,
thepostudy
the study assessed the transmission dynamics of ARV infections and investigated the
assessed
the
transmission
dynamics
of
ARV
infections
and
investigated
the
potential
risk
tential risk factors associated with its spread in poultry farms.
factors associated with its spread in poultry farms.
Viruses 2023, 15, 1318
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In accordance with previous research with similar objectives [10–14], the IDEXX REO®
kit was employed as the sole method for serology testing in the current study, due to its
widespread use in other countries. Although the ELISA has been shown to be a more
sensitive, faster, and more cost-effective method than the existing antibody assay technique
in the viral neutralization of AGP, it has certain limitations [15]. Furthermore, according to
the manufacturer of the ARV ELISA IDEXX kit, the vaccines used for ARV in Morocco are
all detected by this kit, namely Nobilis ReoInac (MSD), Nobilis Reo-S(MSD) and Poulvac
Tri-Reo (Zoetis); the first one is composed based on two ARV strains: 1733 and 2408, the
second one contains the 1133 ARV strain, and the third contains 1133, 2408, and 3005 strains
(the whole virus were used for these vaccines).
For instance, the ELISA method cannot detect all Reovirus strains and serotypes, which
may result in false-negative results. Therefore, it is important to consider the limitations of
this technique when interpreting the results of the current study. It should also be noted
that negative results obtained using a commercial ELISA kit cannot completely rule out the
presence of anti-Reovirus antibodies [15].
The primary objective of this study, conducted in 2021 and 2022, was to investigate
the prevalence of ARV infections in breeder and broiler flocks in six different regions of
Morocco (Casablanca-Settat, Rabat-Salé-Kénitra, Tanger-Tétouan-Al Hoceïma, MarrakechSafi, Fez-Meknès, Oriental). A total of 826 serum samples were collected from 36 flocks
for serological testing, out of which 14 flocks were unvaccinated against ARV, while the
remaining flocks had received ARV vaccination. An indirect ELISA commercial kit was
used for the testing of serum samples. The results revealed that 782 serum samples were
positive for ARV-specific antibodies, indicating a high prevalence (94.6% ± 0.7) of ARV
infections in breeder and broiler flocks across all regions during 2021 and 2022. The high
prevalence of ARV infections in all the examined flocks suggests that ARV infections are
common in poultry farms in Morocco. In the 782 serum specimens found to be positive,
antibody titers ranged from 403 to 22,372, with the average being 5320.8.
Based on the vaccination status, the prevalence of ARV infection was higher in nonvaccinated chickens (98%) compared to vaccinated chickens (92.8%), indicating that the
seroprevalence rate is related to the effectiveness of vaccination against ARV in different
flocks, which can be explained by the involvement of multiple factors. These factors include
the effect of the surrounding environment, the quality of vaccination, which depends on
the techniques used to administer the vaccine, such as the equipment used and the skill
of the person administering it, and the vaccination program itself, which includes the
type of vaccine used, the age at which prophylaxis is administered, and the method of
vaccination. According to several studies, the success of vaccination is influenced by
various factors, including careful storage conditions (maintaining temperature at +2 ◦ C
or +8 ◦ C, protected from light for up to 2 years, and prompt use after reconstitution as
per instructions), the appropriate dosage and administration route, vaccination timing
(administering the primary vaccine between the 7th and 10th week for broiler breeders,
and a booster vaccine before the onset of lay to optimize the passive immunization of
chicks), vaccination coverage and compliance (ensuring complete coverage to minimize
gaps in the vaccination process and protect all individuals), and considering the potential
immunosuppressive effects (avoiding the concurrent use of the S1133 vaccine strain with
live Marek and Gumboro vaccines). Failure to adhere to each of these parameters can lead
to a lower seropositivity rate, even if the flock is considered vaccinated [16–18]; additionally,
it was observed that all samples of unvaccinated 1-day-old broilers tested positive for ARV
infection, with higher mean antibody titers (7458.7 ± 666.9) than unvaccinated broiler
breeders (5149.2 ± 229.1). This could be attributed to the fact that the immune system
of young chickens is not fully developed, and they are more susceptible to infections.
Furthermore, protection against reovirus involves the maternal transfer of antibody through
the yolk to the progeny; this means that the breeders are either vaccinated or have had
contact with the wild ARV during their laying period [19]. On the other hand, older
Viruses 2023, 15, 1318
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chickens have developed age-related resistance to the virus, which could explain the lower
antibody titers observed in the unvaccinated broiler breeders.
The geographical location effect was investigated, and the results indicated that the
prevalence of reovirus was significantly higher in medium-density flocks as compared to
low-density and high-density flocks. Specifically, 98.5% of the sampled birds from mediumdensity flocks tested positive for the virus, whereas only 92.5% and 88.5% of the sampled
birds from low-density and high-density flocks, respectively, were positive for reovirus.
The increased prevalence of the virus in medium-density flocks, particularly among the
non-vaccinated flocks, could be attributed to the higher likelihood of virus transmission
and infection in such flocks. This is due to the close proximity of birds in medium-density
flocks, which can facilitate the spread of the virus through contact, airborne particles, or
contaminated materials.
The seroprevalence of ARV infection was not significantly different among the different regions studied. The highest seroprevalence was observed in Rabat-Salé-Kénitra, where
all of the sampled flocks were positive for ARV antibodies. In contrast, the lowest seroprevalence was observed in Casablanca-Settat, with only 88.5% of the sampled flocks being
positive for ARV antibodies. For the other regions, the seroprevalence ranged from 90% to
97%, with 98.5% for Marrakech-Safi, 97.2% for Fez-Meknès, 95.8% for Tanger-Tétouan-Al
Hoceïma, and 90.8% for Oriental. This indicates that the virus seems to have a relatively
equal presence across all regions, indicating that geographical location does not have a
significant effect on the spread and transmission of the virus.
Regarding periods of collection, the prevalence of ARV specific antibodies was significantly higher in 2021 than in 2022, being, respectively, 97.9% and 91.9%. The higher
seroprevalence observed in 2021 compared to 2022 can be explained by two factors: the
high viral pressure in Moroccan farms due to the importation of day-old breeder chicks of
broiler chickens that were likely already infected with the avian reovirus, and the shortage
of vaccines. The appearance of lameness cases on the Moroccan territory in 2021 was
initially thought to be due to avian reovirus infection, but the PCR results turned out to
be negative. Further investigations revealed that the problem was caused by nutritional
and genetic issues. These findings highlight the need for better biosecurity measures in
the importation of day-old chicks, and the timely provision of sufficient vaccine stocks to
prevent the spread of avian reovirus in Moroccan poultry farms [14].
The serological testing results revealed a high seroprevalence of ARV in unvaccinated
broiler and breeder flocks in Morocco, with values of 100% and 97.4%, respectively, indicating the endemic and circulating nature of this virus within breeder farms, independent
of various factors. The high incidence of ARV infections in breeders is probably due to
two main sources. Firstly, the flocks may become infected by the virus through the environment, as ARVs are commonly found in the poultry population and can be isolated from
the respiratory and gastrointestinal tracts of chickens showing acute or asymptomatic infections [12]. Secondly, contaminated vaccines, especially live-virus vaccines, may represent
another potential source of ARV infections, as there have been reports of occasional avian
reovirus contamination in avian viral vaccines, as reported by Juan Pu, Xingli et al. in their
article [12].
To better understand the circulation of ARV infection in Moroccan poultry farms, we
examined the prevalence of ARV antibodies in unvaccinated broiler breeders of different
age groups. The highest prevalence of ARV antibodies was found in 1-day-old chicks, with
a prevalence of 100%. This suggests that younger chickens have lower immunity due to
their underdeveloped immune system [19]. Chickens in the age group of 21–26 weeks
had the next highest prevalence rate of 99%, followed by chickens in the age group of
8–12 weeks with a prevalence rate of 93.7%. Additionally, the mean antibody titers were
highest at the age of 1 day, at 6288.5 ± 292.7, decreased to 2984.5 ± 230.6at the age of
8–12 weeks, and then increased again to 8175.4 ± 341.1. This trend may be explained by
prolonged exposure and recurrent infections.
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The vaccinated breeder flocks displayed a similar pattern to the unvaccinated flocks,
with a high seroprevalence rate of 98.1% at 1 day of age. This can be attributed to the
vaccination of the parent birds before the chicks are imported from foreign countries. The
seroprevalence rate then decreased to 81.1% at 8–12 weeks of age, which can be explained by
the vaccination schedule that might not have been enough to sustain high antibody levels
at this age. The increase in seroprevalence to 98.9% for the age group of 21–26 weeks can be
explained by the close contact between birds or the transfer of birds to production facilities,
which is a common practice at this stage, and may increase their exposure to environmental
stressors suppressing the immune system, enhancing the exposure to the virus.
The prevalence of ARV infection in the poultry population has been investigated in
several countries. Comparable findings were reported in Romania, by Botuş et al. [20], Iran, by
Bokaei et al. [21], and Canada, by Nham et al. [11], where the seroprevalence rates were 99.5%,
98.3%, and 90.5%, respectively. However, lower seroprevalence rates were recorded in Turkey
(75.9%), by Erol et Şengül [22], Vietnam (85.2%), by Thu H. T. V. [13], and Egypt (80.6%), by
Al-Ebshahy et al. [10]. In contrast, some countries, such as Nigeria [23] and Bangladesh [24],
reported relatively lower prevalence rates of ARV infection in broilers or breeders with
41% and 39.5%, respectively. In India, a seroprevalence rate of 8.7% [14] was reported
in unvaccinated broiler breeder flocks. These findings indicate that ARV infection is
widely prevalent in the poultry population worldwide, with varying degrees of prevalence
across different countries. It underscores the need for continuous monitoring and the
implementation of effective control measures to mitigate the impact of ARV infection on
poultry production.
The variations in the observed seroprevalence rates across different countries suggest
the potential influence of multiple factors, including differences in management practices,
vaccination schedules, environmental conditions, and potential exposure to other viral and
bacterial infections [14]. Additionally, the age of the flock at the time of transport, immune
exposure, and the level of viral excretion may also contribute to these variations. Therefore,
further research is needed to identify the contributing factors and develop effective control
strategies to prevent the spread of ARV globally. The need for such control measures is
especially crucial in countries where high prevalence rates have been reported, such as
Morocco. The development of effective control strategies and a better understanding of the
factors influencing ARV spread would significantly contribute to enhancing overall poultry
health and productivity.
The epidemiology of ARV infections in North Africa is not well understood, particularly in Algeria, Mauritania, and Tunisia, which have shared a molecular study of ARV [25]
but not a serology study. The lack of information on the presence and prevalence of ARV in
these countries makes it difficult to determine the extent of the disease in the region. Since
Morocco has a high incidence of ARV infections, it is crucial to conduct similar studies in
neighboring countries to gain a more comprehensive understanding of the regional context
of ARV in North Africa.
Although our study on reovirus seroprevalence in poultry shares similarities with
other research, there are notable differences in the study design and population that
complicate comparisons. These differences include the criteria for defining flock status as
positive or negative, the antibody titer thresholds used to indicate reovirus seropositivity,
the age of broilers and breeders during sampling, and the vaccination status of breeders [11].
These dissimilarities may have influenced the results and should be considered when
interpreting findings from various studies. For instance, the threshold for classifying a flock
as seropositive may differ depending on the ELISA kit used or the sensitivity of the test.
Additionally, the age of broilers during sampling can affect seroprevalence rates, with older
birds potentially having higher antibody titers due to prior exposure and close contact [11].
Vaccination status can also impact seroprevalence rates, with vaccinated flocks potentially
having a high seroprevalence rate due to their prior immunization.
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5. Conclusions
The findings of this study provided valuable insights into the control and management of ARV infections in poultry farms in Morocco. The results served as a foundation
for future research aimed at comparing the efficacy of different vaccination strategies in
controlling ARV infections in poultry farms across the country; focusing on evaluating
the long-term effectiveness of different ARV vaccines, and identifying the most suitable
vaccination schedules for different age groups and breeds of poultry; and investigating the
potential benefits of combining vaccination with other control measures, such as biosecurity
protocols, to enhance the efficacy of ARV control programs. This study will also contribute
to filling the current knowledge gaps regarding ARV infections in chickens in Morocco,
supporting the sustainable development of the poultry industry in the country.
Author Contributions: A.A. participated in the design of the study, drafted the manuscript, collected
the sera, and analyzed the results. S.F. participated in the design of the study, helped with ELISA, and
corrected the manuscript. A.Z. and N.A. carried out the serological analyses and interpretation, and
corrected the manuscript. M.B. and A.F. helped with analyzing the data and statistical analyses. M.M.
collected the sera and helped with the draft of the manuscript. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Avian Pathology Unit at the Agronomy and Veterinary
Hassan II (Grant 1612) and Cabinet Tit Mellil.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank the partners and laboratory technicians for their help with this project.
Conflicts of Interest: The authors declare no conflict of interest.
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