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Rev. sci. tech. Off. int. Epiz., 2011, 30 (3), 931-937
Ornithobacterium rhinotracheale
and Mycoplasma synoviae
in broiler chickens in Jordan
D.A. Roussan (1)*, R.H. Al-Rifai (2), G.Y. Khawaldeh (1), W.S. Totanji (1)
& I. Shaheen (1)
(1) Technical Department, Provimi, Jordan, P.O. Box 499, Amman 11118, Jordan
(2) Department of International Health Development, Division of Public Health, Tokyo Medical and Dental
University, Tokyo, Japan, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
*Corresponding author: [email protected]
Submitted for publication: 30 August 2010
Accepted for publication: 24 June 2011
Summary
A cross-sectional study was conducted from November 2008 to July 2010 in
commercial broiler flocks in southern (n = 50) and northern (n = 50) areas of
Jordan, to determine the flock-level prevalence of Ornithobacterium
rhinotracheale (ORT) and Mycoplasma synoviae (MS) infections. Tracheal
swabs were collected from commercial broilers with respiratory disease and
tested by polymerase chain reaction. In total, 21% (95% CI: 18–45%) and 25%
(95% CI: 20–51%) of commercial broiler flocks were positive for ORT and MS,
respectively. In the southern areas the prevalence of flocks with positive
tracheal swabs for ORT and MS was 16% and 10%; in the northern areas the
prevalence was 26% and 40%, respectively. Of the flocks tested, 7% were
infected with ORT and MS simultaneously. Further epidemiological studies are
recommended to determine risk factors and evaluate the economic
consequences of ORT and MS infections in the region. Furthermore, studies are
required to isolate ORT and MS and develop vaccines against the local field
isolates.
Keywords
Broiler – Jordan – Mycoplasma synoviae – Ornithobacterium rhinotracheale –
Polymerase chain reaction.
Introduction
environmental factors, the immune status of the flock, and
the presence of other infectious agents (23).
Ornithobacterium rhinotracheale (ORT) is a recently
described species of bacterium (24) associated with
respiratory disease, growth retardation, mortality and
decreased egg production in chickens and turkeys (7). It
can cause a highly contagious disease in poultry, although
the severity of clinical signs, duration of the disease and
mortality have been found to be extremely variable (23).
The bacterium has been isolated from chickens, turkeys,
chukar partridges, quail, ducks, geese, ostriches and
guinea fowl. It can be a primary or secondary aetiological
agent, and this depends on strain virulence, adverse
Mycoplasma synoviae (MS) is an important pathogen of
domestic poultry, and causes economic losses to the
poultry industry (14). The agent most frequently causes a
subclinical upper respiratory infection, which can progress
to respiratory disease with air sac lesions when exacerbated
by other respiratory pathogens (e.g. Newcastle disease
virus, infectious bronchitis virus) or when more virulent
strains of MS are involved (15, 17). The infection can also
become systemic and this results in an acute or chronic
infectious synovitis (14).
932
In Jordan, where the poultry industry is of great
importance both socially and economically, disease
prevention programmes are crucial to avoid virus spread
and the development of disease (12). Respiratory diseases
in chickens in Jordan are a major concern, because they
cause catastrophic economic losses to the poultry industry.
No previous studies have been carried out in Jordan to
determine the involvement of ORT and MS in respiratory
disease in broiler chicken flocks. The aim of the present
study was to estimate the flock-level prevalence of ORT
and MS infections in broiler flocks located in the southern
and northern areas of Jordan, using polymerase chain
reaction (PCR) on tracheal swab samples.
Materials and methods
Broiler flocks
This study, which was carried out between November
2008 and July 2010, involved the examination of 100
commercial broiler chicken flocks, located in the southern
(n = 50) and in the northern (n = 50) areas of Jordan, in
which chickens were identified as suffering from
respiratory disease. The chickens were kept until they were
40 to 45 days old and then sent for processing. The broiler
chicken vaccination programme usually involved two live
vaccines against Newcastle disease virus and infectious
bronchitis virus, administered either by spray or in the
drinking water at 1 and at 11 to 12 days old, and one
infectious bursal disease vaccine (mild or moderate strain)
administered in the drinking water at 12 days old.
The southern and northern areas of Jordan are the most
important parts of the country for the poultry industry.
Taken together, these areas contain 170 broiler-type
chicken farms, with an average of 10,000 to 20,000
chickens per farm (12). The flocks were selected with the
help of the Ministry of Agriculture, which maintains
records of the name of the owner and the location of each
broiler flock in these regions. In the majority of flocks,
signs of respiratory disease usually appeared in chickens of
29 to 34 days old; six chickens from each flock were taken
during the acute phase of disease and sent to the Provimi
Jordan laboratory. Affected chickens suffered from nasal
and ocular discharge and conjunctivitis, as well as being
weak and reluctant to move. Gross lesions observed in
these flocks included a mild congestion of the trachea and
mild airsaculitis. In the laboratory, samples of tracheas
from each flock were collected and stored at –80°C until
used for DNA extraction. None of these flocks was
vaccinated against MS or ORT.
Deoxyribonucleic acid extraction
Frozen tracheas from each flock were swabbed with sterile
swabs (Heinz Herenz Medizin GmbH, Hamburg,
Rev. sci. tech. Off. int. Epiz., 30 (3)
Germany). The swabs were placed in 1,000 µl phosphatebuffered saline (PBS; pH 7.2) and were scraped on the side
of the tube to facilitate removal of the contents from the
swab head. Bacterial DNA was extracted and purified using
a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA)
according to the manufacturer’s recommended procedure.
Molecular polymerase chain reaction
to detect Ornithobacterium rhinotracheale
and Mycoplasma synoviae
The primers used in this study for ORT consisted of the
following sequences: OR16S-F1 (5´-GAG AAT TAA TTT
ACG GAT TAA G-3´) and OR16S-R1 (5´-TTC GCT TGG
TCT CCG AA GAT-3´) (Alpha DNA, Montreal, QC,
Canada), which amplify a 784-base pair (bp) fragment of
the 16S ribosomal ribonucleic acid (rRNA) gene of ORT
but not of other closely related bacteria (11). The primers
used for MS consisted of the following sequences: MS-F
(5´-GAG AAG CAA AAT AGT GAT ATC A-3´) and MS-R
(5´-CAG TGG TCT CCG AAG TTA ACA A-3´) (Alpha
DNA, Montreal, QC, Canada), which are complementary
to the 16S rRNA gene of MS, and the PCR produces a 214bp fragment in positive samples (16). The PCR mix was
prepared in a volume of 50 µl containing 25 µl of master
mix (Taq polymerase 5 U/µl, 400 mM of deoxynucleotide
5´-triphosphate mixture, and 3 mM of MgCl2), 18 µl of
nuclease-free water (Promega Corp., Madison, WI, USA),
1 µl (50 pmol/µl) of each oligonucleotide, and 5 µl of DNA
template. The samples were amplified under the following
conditions and the reaction was carried out in a DNA
Engine® thermal cycler (Bio-Rad Laboratories Ltd,
Mississauga, ON, Canada). For each flock there were two
PCR tubes, one tube for ORT and one for MS, which were
included in the same run with the same programme but
different annealing temperatures (the PCR tubes for ORT
and MS were placed in specific wells according to the
annealing temperature). The temperature gradient
programme for one cycle consisted of 3 min at 94°C, then
35 cycles at 94°C for 2 min (denaturation), 58°C for 1 min
(for ORT), 54°C for 1 min (for MS; annealing), and 72°C
for 2 min (extension). Ornithobacterium rhinotracheale
vaccine Nobilis OR inac (Intervet, Wim de Körverstraat,
AN Boxmeer, the Netherlands) and MS vaccine Vaxsafe
MS® (Bioproperties, Australia) were used as positive
controls for DNA extraction and PCR. A negative control
(nuclease-free water) was also used in each run.
Agarose gel electrophoresis
Polymerase chain reaction products were electrophoresed
on a 2% agarose gel in Tris-acetate-EDTA buffer (40 mM of
Tris and 2 mM of ethylene-diamine-tetra-acetic acid
[EDTA], pH 8.0) containing ethidium bromide (Promega
Corp., Madison, WI, USA) for 45 min at 100 V and
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Rev. sci. tech. Off. int. Epiz., 30 (3)
visualised under UV light (AlphaImager, AlphaInnotech,
San Leandro, CA, USA).
Statistical analysis
Confidence limits (95%) for prevalence were calculated
using an approximation to the standard normal
distribution. The unit of statistical analysis was the flock.
Comparison of prevalence between the two areas of
Jordan was done using the chi-square test, and p-values of
≤0.05 were considered statistically significant. All analysis
was performed using SAS Version 6.0 (20).
Results
Samples from a total of 100 broiler flocks with a history of
respiratory disease were received by the Provimi Jordan
laboratory from southern (n = 50) and northern (n = 50)
areas in Jordan and tested by PCR for the presence of ORT
and MS in tracheal samples. Of the 100 flocks studied,
21% (95% CI: 18–45%) and 25% (95% CI: 20–51%) were
positive for ORT and MS infections, respectively. The
distribution of flocks in the southern and northern areas of
Jordan that were positive by PCR is shown in Table I. In the
southern areas the prevalence of flocks with positive
tracheal swabs for ORT and MS was 16% and 10%,
respectively; in the northern areas the prevalence was 26%
and 40%, respectively; the difference between the regions
was statistically different (X2 = 4.94, d.f. = 1, p = 0.0263).
In addition, 7% of the 100 flocks tested were infected with
ORT and MS simultaneously.
Discussion
Respiratory tract infection is a major problem in the poultry
industry throughout the world. It has a major economic
impact as a result of treatment costs and losses caused by the
deaths of animals and higher rates of carcass condemnation.
Respiratory diseases in poultry arise from various causes,
including bacteria (5), viruses (2) and fungi (1).
In the current study, ORT and MS were detected by PCR in
21 and 25 of the flocks tested, respectively (Table I). Given
that all the birds tested in this study were older than
four weeks of age, were suffering from respiratory disease,
and were not vaccinated against MS and ORT, these data
suggest that the infected flocks were exposed in the field to
MS and/or ORT. Respiratory diseases in poultry have been
reported to be caused by mixed or single infections with
several agents (18, 19, 25, 27). In the current study, 7% of
the flocks tested were infected with ORT and MS at the
same time.
Ornithobacterium rhinotracheale infections have been
reported in many countries around the world, including
Belgium (26), Canada (13), the United States (6), Israel
(4), the Republic of Korea (28), Japan (22), Egypt (8),
Turkey (10), Chinese Taipei (21) and Iran (3). In Jordan,
ORT was first isolated from broilers in 2001 (9). In the
current study, ORT was detected in 21 out of the 100 flocks
tested. Of these 100 flocks, 14% were diagnosed to be
infected with ORT alone, whereas in the other 7% of flocks
ORT was combined with MS (see Table I). This study and
previous reports (9) have confirmed the endemic nature of
ORT infection in Jordan.
Table I
Distribution of Ornithobacterium rhinotracheale and Mycoplasma synoviae infections detected by polymerase chain reaction in 100
commercial broiler flocks in the southern (n = 50) and northern (n = 50) areas of Jordan
PCR-positive flocks
Number
Percentage
PCR-negative flocks
Number
Percentage
Area
Agent
Southern
ORT alone
6
12
44
88
MS alone
3
6
47
94
MS + ORT
2
4
48
96
ORT total
8
16
42
84
MS total
5
10
45
90
ORT alone
8
16
42
84
MS alone
15
30
35
70
MS + ORT
5
10
45
90
ORT total
13
26
37
74
MS total
20
40
30
60
Northern
MS: Mycoplasma synoviae
ORT: Ornithobacterium rhinotracheale
PCR: Polymerase chain reaction
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Rev. sci. tech. Off. int. Epiz., 30 (3)
Mycoplasma synoviae was detected in 25 of the 100 flocks
tested. Eighteen percent were diagnosed to be infected
with MS alone, whereas in the other infected flocks MS was
combined with ORT (Table I). Mycoplasma synoviae has
been reported to occur throughout the world (14). This is
the first report of the involvement of MS in respiratory
disease seen in broiler flocks in Jordan, and it will pave the
way for further studies of MS.
countermeasures. Furthermore, studies are required to
isolate ORT and MS and to develop vaccines against the
local field isolates. In addition, farmers need to be
educated about the signs of infection and the importance
of these pathogens.
The high prevalence of both pathogens in the northern
area in comparison with the southern area may be
attributed to the diversity of the chicken population
(layers, breeders and broilers) and the presence of a large
number of broiler farms in the northern area in
comparison with the southern area.
This study was funded by Provimi Jordan. Deepest
gratitude is also extended to all members of Provimi
Jordan.
Acknowledgements
Conclusion
This study has shown that ORT and MS are involved in
respiratory disease in broiler chickens in Jordan. Further
studies are necessary to assess the circulating strains, the
economic losses caused by infections and co-infections of
MS and ORT, and the costs and benefits of
Infections à Ornithobacterium rhinotracheale
et à Mycoplasma synoviae chez des poulets de chair en Jordanie
D.A. Roussan, R.H. Al-Rifai, G.Y. Khawaldeh, W.S. Totanji & I. Shaheen
Résumé
Une étude transversale a été conduite de novembre 2008 à juillet 2010 dans des
élevages commerciaux de poulets de chair dans des zones situées au sud
(n = 50) et au nord (n = 50) de la Jordanie afin de déterminer la prévalence au
niveau des élevages des infections à Ornithobacterium rhinotracheale et à
Mycoplasma synoviae. Des écouvillons trachéaux prélevés chez des volailles
commerciales présentant des troubles respiratoires ont été soumis au test
d’amplification en chaîne par polymérase (PCR). Les résultats ont été positifs
pour O. rhinotracheale et M. synoviae dans respectivement 21 % (intervalle de
confiance [IC] à 95 % compris entre 18 % et 45 %) et 25 % (IC à 95 % compris
entre 20 % et 51 %) des élevages commerciaux de poulets de chair.
935
Rev. sci. tech. Off. int. Epiz., 30 (3)
La prévalence des élevages possédant des volailles dont les écouvillons
trachéaux se sont révélés positifs pour O. rhinotracheale et M. synoviae était
respectivement de 16 % et de 10 % dans les zones du Sud couvertes par l’étude,
et de 26 % et 40 % dans les zones du Nord. Parmi les élevages analysés, 7 %
présentaient une infection concomitante à O. rhinotracheale et à M. synoviae.
Des études épidémiologiques plus approfondies permettront d’élucider les
facteurs de risque et d’évaluer les conséquences économiques de ces
infections dans la région. En outre, des études devront être conduites pour isoler
O. rhinotracheale et M. synoviae et pour mettre au point des vaccins adaptés
contre les souches de terrain isolées localement.
Mots-clés
Amplification en chaîne par polymérase – Jordanie – Mycoplasma synoviae –
Ornithobacterium rhinotracheale – Poulet de chair.
Ornithobacterium rhinotracheale y Mycoplasma synoviae
en pollos asaderos de Jordania
D.A. Roussan, R.H. Al-Rifai, G.Y. Khawaldeh, W.S. Totanji & I. Shaheen
Resumen
Los autores describen un estudio transversal realizado entre noviembre de 2008
y julio de 2010 con pollos asaderos de granjas industriales de las zonas
meridional (n = 50) y septentrional (n = 50) de Jordania con objeto de determinar
la prevalencia en las bandadas de las infecciones por Ornithobacterium
rhinotracheale (ORT) y Mycoplasma synoviae (MS). Tras extraer hisopados
traqueales de pollos con afección respiratoria, se analizaron esas muestras por
reacción en cadena de la polimerasa (PCR). En total, un 21% (IC 95%: 18–45%) y
un 25% (IC 95%: 20–51%) de las bandadas industriales resultaron positivas para
ORT y MS, respectivamente. En las zonas meridionales, la prevalencia de
bandadas con muestras traqueales positivas para ORT y MS fue del 16% y el
10%, respectivamente; en las zonas septentrionales, esos porcentajes fueron del
26% y el 40%. De las bandadas analizadas, un 7% estaban infectadas a la vez por
ORT y MS. Los autores recomiendan que se efectúen nuevos estudios
epidemiológicos para determinar los factores de riesgo y evaluar las
consecuencias económicas de ambas infecciones en la región. También se
precisan estudios que permitan aislar ambos microorganismos y elaborar
vacunas contra las cepas locales aisladas.
Palabras clave
Jordania – Mycoplasma synoviae – Ornithobacterium rhinotracheale – Pollo asadero –
Reacción en cadena de la polimerasa.
936
Rev. sci. tech. Off. int. Epiz., 30 (3)
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