Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
192
ISOLATION OF NEWCASTLE DISEASE VIRUS (NDV) IN
EMBRYONATED CHICKEN EGGS
Firas T. Mansour; Hazim T. Thwiny; Khadeeja S. Madhi; Surra R .Khamees
Department of Microbiology, College of Veterinary Medicine, University of
Basrah,Basrah,Iraq
Key words: Newcastle disease virus, vaccine, hemagglutination
ABSTRACT
Newcastle disease virus (NDV) strains have been divided into three groups: virulent
(velogenic), moderately virulent (mesogenic), and non-virulent strains (lentogenic). The nonvirulent
virus strain (LaSota strain) has been used as a live vaccine, which gives good immunity
against the virulent strains. The aim of the study was to grow and propagate Newcastle disease
virus in the lab, determination ofcytopathic effects in chicken embryos, and confirmation of
virus growth by hemagglutination test. Non-virulent strain (LaSota strain)represented by live
vaccine was used for this purpose. Embryonated eggs were inoculated with the virus and
incubated for 48 hours; and theallantoic fluids were then collected for further processing.
Petichial hemorrhages were clearly observed in the embryos following infection while in the
un-inoculated eggs; the embryos appeared normal and did not show any lesions. For further
virus growth confirmation, the presence of virus in the allantoic fluid was determined by
hemagglutiation test. This finding is considered as a starting point for Newastle disease virus
antigen preparation, which is essential for the applications of several laboratory techniques.
INTRODUCTION
Newcastle disease (ND) is one of the most significant diseases of poultry worldwide. It is
caused by Avian Paramyxoviruses type-1 (APMV-1) which is classified with the other avian
paramyxovirusesin the genus Avularius, family Paramyxoviridae(1). Newcastle disease virus
(NDV) strains have been divided into three groups: virulent (Velogenic), moderately virulent
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
193
(Mesogenic) and non-virulent (Lentogenic), which differ in the number of basic amino acids at
the cleavage site of the fusion protein(2,3). Signs of infected birds are very variable depending
on the strain of virus,species of bird,concurrent disease and preexisting immunity(4,5).
The lentogenic(LaSota vaccine) strains of low virulence are commonly used worldwide, and
can provide protection against virulent strains if the vaccines are viable, administered correctly
to healthy birds (6).
The disease can be diagnosed in the laboratory using some techniques such as enzyme linked
immunosorbent assay(ELISA), polymerase chain reaction (PCR) and gene sequencing.The
most convenient method of propagating Newcastle disease virus in the laboratory is by the
inoculation of the allantoic cavity of embryonated eggs with the clinical samples. This method
is also important to grow the virus for other purposes such as preparation of viral antigens and
vaccine production(7).
The hemagglutination assay (HA) is a most common indirect method to quantify amounts of
virus particles, generated from cell culture supernatant or allantoic fluid harvested from chicken
eggs. This assay relies on the fact that many viruses contain proteins that can bind and
agglutinate red blood cells (8).
The aim of this study was to propagate a low virulence strain of Newcastle disease virus
(represented byLaSota vaccine) in embryonated chicken eggs as a starting point for virus
antigen preparation.
MATERIALS AND METHODS
Virus propagation and harvesting
Viruses used in this study were propagated in fertile chicken’s eggs. Working virus
concentration was prepared by diluting the stock virus (vaccine) 1:2000 in phosphate buffer
saline (PBS) containing 1% antibiotics (100 U/ mL penicillin and 100 ug/ mL streptomycin).
The fertilized eggs were incubated for 9 days at 37.5˚C and then inoculated with the virus. At
the end of this period of incubation, the embryos were candled using an egg-candling box in a
dark room and the air sacs were outlined with a pencil in order to determine the site for
injection. Eggs without developing embryos were discarded. After wiping the egg surface with
ethanol, a small hole was made in the shell at the site of injection with a specific drill without
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
194
damaging the shell membranes. A hypodermic syringe (1 mL) fitted with a fine needle was
used for virus inoculation. The needle was passed through the hole in the egg shell, through
chorioallantoic membrane, and the virus (0.1 mL) was injected in the allantoic cavity, which is
filled with allantoic fluid. The hole was carefully sealed with wax or tape, and the eggs were
placed at 37.5˚C for 48 hr.
Figure 1: Inoculation of Newcastle disease virus into a chicken embryo.
A healthy 8 day old embryo is represented schematically; 0.1 ml of virus was inoculated
through the air sac into the allantoic cavity as indicated with a syringe.
Allanoic fluid was collected from eggs, 48 hours post inoculation. Embryos were put in clean
Petri dishes to observe the cytopathic effects. Photos were then taken for documentation.
Virus detection by hemagglutination
The allantoic fluid was processed for hemagglutination assay to detect the viruses.
Blood samples were collected from chicken and placed in tubes coated with anticoagulant
layer. Samples were first centrifuged and the serum was discarded. RBCs were purified using
Alsever’sbuffer to obtain 5% pure RBCs following series of centrifugations.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
195
The presence of viruses in the allantoic fluid was confirmed using the hemagglutination assay.
Briefly, a ceramic agglutination plate which contains six wells were cleaned and prepared for
this experiment. A drop of allantoic fluid was mixed with another drop of RBC in the well.
The negative control was represented by mixing a drop of RBC with another drop of Phosphate
Buffer Saline (PBS). The reaction was left for a few minutes and photos were taken for
documentation.
RESULTS
Figure 2 Chicken embryos inoculated with Newcastle disease virus.
Embryopathy in a 10 day old chicken embryo inoculated with Newcastle disease virus
vaccine strain showing petechial hemorrhages (indicated by arrows). Uninfected
embryos showed no any lesions.
Detection of viruses by hemagglutination
Results of hemagglutinationtestofallantoic fluid,which has been collected from inoculated eggs
were positive, which confirms the growth of the virus. Negative result was shown in the control
group(Figure 3).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
196
Figure 3: Detection of virus growth by hemagglutination test.
The positive hemagglutination result is clearly observed in the allantoic fluid of
inoculated eggs while in the non-inoculated, there is no evidence of hemagglutination.
DISCUSSION
Newcastle disease virus (NDV) is an ideal virus system for elucidating determinants of viral
pathogenicity. Some strains of this virus are important agricultural pathogens that cause disease
in poultry with a high mortality while other strains are avirulent (such sLaSota strain) and used
as vaccines. Methods for the preparation and titration of virus stocks are essential for all of
these purposes. The use of embryonated chicken eggs to grow the virus is considered as a
superior method for virus isolation (9).
In this project, an avirulent virus vaccine strain was successfully isolatedinembryonated
chicken eggs to produce a high titer of virus stock which is essential for virus antigen
preparation. Such virus antigens are important in performing various laboratory techniques
such as ELISA, hemagglutination, heamagglutination inhibition and neutralization tests.
The Lasota virus strainhas been grown in embryonated eggs for the purpose of vaccine
production (7). In comparison, in the present study, this strain was grown for another purpose
which is a local antigen preparation. In this regard, it is strongly recommended to prepare
antigens from the isolated viruses using different laboratory methods. Furthermore, it is crucial
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
197
to decide which viral antigen (protein) should be isolated(e.g the type of antigen used in ELISA
test is different from that used for hemagglutination test).
The kinds of approaches outlined above may lead to the potentially new findings, which will
help to prepare different kinds of antigens necessary for a variety of laboratory techniques, and
hence warrants further studies.
عزل فایرس مرض النیوکاسل من أجنة الدجاج
فراس طعمھ منصور ،حازم طالب ثوینی ،خدیجھ مھدی ،سرى خمیس
فرع الاحیاء المجھریھ ،کلیة الطب البیطری وجامعھ البصره ، البصره ،العراق
الخلاصة
تقسم سلالات فیروس مرض نیوکاسل إلى ثلاثة مجموعات: سلالات عالیة الضراوة، معتدلة الضراوة، وغیرضاریة. للحد
والذی یمثل السلالة غیر الضارة ولکنھا بنفس الوقت تعطی مناعة ضد LaSota من الاصابة بمرض النیوکاسل یستخدم لقاح
السلالات عالیة الضراوة. الھدف من ھذه الدراسة ھو تنمیة فیروس النیوکاسل فی المختبر، وتحدید الافات المرضیة
المصاحبة للاصابة بالفیروس فی أجنة الدجاج، وتأکید نمو الفیروس عن طریق اختبار التلازن الدموی. تم استخدام سلالة
لھذا الغرض متمثلة باللقاح الحی الذی یستخدم فی التحصین الحقلی ضد المرض. تم حقن (LaSota غیر ضاریة (سلالة
بیض الدجاج المخصب بالفیروس ومن ثم حضنھا لمدة 48 ساعة، بعدھا تم جمع السوائل الموجودة فی داخل البیضة. لوحظ
نزیف نقطی بوضوح فی الأجنة بعد الإصابة فی حین أن الاجنة فی البیض غیرالمحقون بالفیروس کانت طبیعیة ولم تظھر
فیھا ای افات مرضیة. لمزید من التأکید لنمو الفیروس، تم تحدید وجود الفیروس فی سوائل البیضة عن طریق اختبار التلازن
الدموی. وتعتبر ھذه النتیجة بمثابة نقطة انطلاق لإعداد مستضدات لفیروس النیوکاسل والذی یعد أمر ضروری لتطبیق عدة
تقنیات فی مختبر علم الفیروسات.
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