MOLECULAR CHARACTERIZATION OF HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES CIRCULATING IN COMMERCIAL BROILER CHICKENS IN SOME LOCALITIES IN SOHAG PROVINCE | |||||||||||||||||||||||||||||||||||||||||||||||
Assiut Veterinary Medical Journal | |||||||||||||||||||||||||||||||||||||||||||||||
Article 1, Volume 58, Issue 133 - Serial Number 2, April 2012, Page 1-13 PDF (2.47 MB) | |||||||||||||||||||||||||||||||||||||||||||||||
Document Type: Research article | |||||||||||||||||||||||||||||||||||||||||||||||
DOI: 10.21608/avmj.2012.172806 | |||||||||||||||||||||||||||||||||||||||||||||||
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Authors | |||||||||||||||||||||||||||||||||||||||||||||||
M.A. MOHAMED1; S-E. A. SULTAN2; A.I. AHMED3; A.M. OSMAN4 | |||||||||||||||||||||||||||||||||||||||||||||||
1Department of Poultry Diseases, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt. | |||||||||||||||||||||||||||||||||||||||||||||||
2Department of Microbiology (Virology), Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. | |||||||||||||||||||||||||||||||||||||||||||||||
3Department of Poultry Diseases, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt | |||||||||||||||||||||||||||||||||||||||||||||||
4Department of Poultry Diseases, Faculty of Veterinary Medicine, Sohag University, Sohag, Egypt | |||||||||||||||||||||||||||||||||||||||||||||||
Abstract | |||||||||||||||||||||||||||||||||||||||||||||||
Thirty one tracheal swabs were collected from diseased broiler chickens experiencing respiratory signs and congestion of head and shank regions and molecularly characterized in Sohag province. The results showed, twenty five (80.6%) of swabs were confirmed positive for carrying type A Avian influenza virus (AIV) by using rapid antigen detection kit. Total RNAs were extracted from the 17 positive AI type A swabs and One step RT-PCR test was performed for subtyping using specific primers targeting H5, H7, N1 and N2. AIV H5 subtype was detected in 9 samples and the rest was untypeable with H7 primers. From the 9 H5 strains, one H5N1 detected and 3 were H5N2 that reflects the presence of two subtypes co-circulated at that time of infection and other untypeable strains with our available primers. Molecular pathotyping was carried on the fully characterized H5 strains using Restriction Enzyme Cleavage Pattern (RECP) assay with the aid of MboII restriction enzyme as a new rapid method to avoid the disadvantages of reliable conventional methods (pathogenicity index (IVPI) in specific pathogen free (SPF) chickens and sequencing of hemagglutinin (HA) gene cleavage site). The results revealed that 3 out of 4 (75%) examined H5N1 and H5N2 viruses were pathogenic and one H5N2 strain was low pathogenic. The SDS-page profiling revealed that the examined field isolates were distinct to two clusters; the first one having structural polypeptide: Haemagglutinin (61 KD) and (89KD) protein and the second cluster contains 61 KD, 89KD protein and 50 KDa neuramindase protein. In addition the western blotting showed that the killed Chinese H5N1 vaccine was used in Sohag province by farmers was of low antigenicity which the antibodies reacted only with haemagglutinin protein (61KDa) in all examined strains and one strain reacted with a high molecular weight protein (89KDa). Our data provide proof for the prevalence of H5N2 subtype than H5N1 as well as untypeable strains and low antigenicity of the used vaccine that reflects the low efficiency of the used vaccine in Sohag. So, the field strains should be updated in a timely manner through surveillance and accompanying laboratory evaluation of contemporary viruses for antigenic similarity with existing vaccine strains and so production of good immunity. | |||||||||||||||||||||||||||||||||||||||||||||||
Keywords | |||||||||||||||||||||||||||||||||||||||||||||||
Keywords; Avian influenza; RT-PCR; rapid pathotyping and antigenic analysis | |||||||||||||||||||||||||||||||||||||||||||||||
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Molecular Characterization of Highly Pathogenic Avian Influenza Viruses Circulating in Commercial Broiler Chickens in Some Localities in Sohag Province
M.A. Mohamed*, S-E. A. Sultan***, A.I. Ahmed** and A.M. Osman****
* Department of Poultry Diseases, Faculty of Veterinary Medicine, AssiutUniversity, Assiut, Egypt. ** Department of Poultry Diseases, Faculty of Veterinary Medicine, SouthValleyUniversity, Qena, Egypt. *** Department of Microbiology (Virology), Faculty of Veterinary Medicine, SouthValleyUniversity, Qena, Egypt. **** Department of Poultry Diseases, Faculty of Veterinary Medicine, SohagUniversity, Sohag, Egypt. Corresponding author: moemenassiut@hotmail.com __________________________________________________________________________________________
Abstract _____________________________________________________________________
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Keywords, Avian influenza, RT-PCR, rapid pathotyping and antigenic analysis __________________________________________________________________________________
التوصيف الجزيئي لفيروسات أنفلونزا الطيور المتداولة في الدجاج اللاحم التجاري من بعض المواقع في محافظة سوهاج
مؤمن عبد العظيم محمد ، سراج الدين احمد سلطان ، أحمد ابراهيم احمد ، احمد محمد عثمان
تم جمع واحد وثلاثون مسحة قصبة هوائية من الدجاج اللاحم المريض والذى کان يعاني من مشاکل تنفسية واحتقان في مناطق الرأس والساق. أظهرت النتائج أن 25 من المسحات (80.6٪) إيجابية لحمل فيروس أنفلونزا الطيور (AIV) من النوع (أ) باستخدام عدة الکشف السريع لمستضد فايروس انفلونزا الطيور. تم استخراج RNAs من مجموع 17 مسحة ايجابية لفيروس انفلونزا الطيور ثم أنجز اختبار الخطوة الواحدة لتفاعل البلمرة العکسية المتسلسل (One Step RT-PCR) وذلک للتصنيف النوعى لفايروس انفلونزا الطيور باستخدام بادئات تستهدف H5، H7،N2 و N1. تم اکتشاف 9 من 17 عينة تحمل H5 AIV والباقي کان غير متعرف عليه (untypeable) مع بادئات H7. من الـ9 سلالات H5، کانت واحدة H5N1وثلاث H5N2 الذي يعکس وجود نوعين في ذلک الوقت من العدوى وغيرها من السلالات الغير معرفة untypeable. وللتعرف على ضرواة معزولات H5 تم استخدام طريقة التعرف الجزيئي على الضرواة باستخدام تقييد نمط الانقسام الانزيمى (RECP) باستخدام انزيم MboII کأسلوب سريع وجديد لتجنب مساوئ الطرق التقليدية الموثوق بها مثل مؤشر المرضية (IVPI) في الدجاج الخالي من مسببات الأمراض (SPF) وتسلسل موقع الانقسام فى جين تلازن الدموية (HA) ، وأظهرت النتائج أن 3 من 4 عترات من H5N1 و H5N2 (75٪) کانت شديدة الإمراض(HPAI) وسلالة واحدة H5N2 کانت منخفضة الإمراض. وکشف التنميط باستخدامSDS-PAGE أن العزلات مجال الفحص کانت مميزة على مجموعتين الأولى تحتوى على: الهيماغلوتينين (61 کيلودالتون) و(89 کيلودالتون) بروتين، والمجموعة الثانية تحتوي على 61 کيلودالتون ، والبروتين 89 کيلودالتون و 50 کيلودالتون (neuramindase). وبالإضافة إلى ذلک أظهر استخدام الغربي النشاف ان السيرم المحصود من الدجاج المحصن بلقاح H5N1 الصيني المستخدم في محافظة سوهاج من قبل المزارعين منخفض الکفاءة نتيجة لتفاعله فقط فى جميع العينات المفحوصة مع المستضداد البروتينى للهيماغلوتينين (61 کيلودالتون) ماعدا فى سلالة واحدة اتحد مع بروتين اضافى عالي الوزن الجزيئي (89 کيلودالتون). من النتائج السابقة نقدم دليلا للانتشارالاکبرلسلالة H5N2 عنها من H5N1 بجانب تواجد انواع اخرى غيرمصنفة مما يفسر انخفاض کفاءة اللقاح المستخدم الى جانب ضعف المستضد البروتينى للتحصين المستخدم. لذلک ينبغي تحديث السلالات المستخدمة فى انتاج اللقاح لتکون معاصرة لسلالات المتدوالة والمعزولة من الوبائيات الحادثة.
Table1: The oligonucleotide sequence of primers used for one step RT-PCR amplification.
Fig.1 (A): Congestion of the comb and Fig.1 (B): Ecchymotic hemorrhage on the shunk periorbital oedema
Fig.2 (A): Showing congestion of the muscles. Fig.2 (B): Hemorrhages on the junction and subcutaneous vessels between proventiculus and gizzard.
Fig.2 (C): Engorgement of duodenal blood vessels
Fig.3: Showing positive reaction of rapid avian Fig.4: RT-PCR analysis of the subtype specific primer influenza antigen test kit of H5.Lane 7-10 amplicons at 219bp indicating positive reaction. Lane 11-12 negative samples. Lane M: 1Kbplus DNA ladder.
Fig.5. Subtyping by RT-PCR using N1 primer, lane Fig. 6. Subtyping of H5 AIV positive samples by M: 1Kb DNA ladder, Lane 14: positive sample RT-PCR using N2 specific primer, Lane 13, Sample for N1 giving amplicon at 616bp and 2, 17 positive lanes for N2 primer giving the same weight of positive control sample. product at 434bp and compatible with positive control sample.
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