Arshad, Siti Suri

Universiti Putra Malaysia Institutional Repository

Agricultural SciencesMicroscopy analysis of trachea and kidney of chicken infected withnephropathogenic Infectious bronchitis virus MH5365/95Sit! Sur! ArohadFacultyof Veterinary MedicineUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaTelephone Number a/Corresponding Author: 03- 89468306E-mailolCorrespondingAuthor:suri@vet.upm.edu.myKey words: kidney, nephropathogenic IBV, scanning electron microscopy, trachea, transmission electronmicroscopy, kidneyIntroductionAvian Infectious Bronchitis virus is common in chickens worldwide. It is belongs to group III coronavirus of the genusCoronavirus of the Coronaviridae family. It was first reported in baby chicks by Schalk and Hawn in 1931, in North Dakota.Although it was thought to be primarily a respiratory disease of chick, avian IBY has since found to be an economicallyimportant disease of adult chicken. The disease has been associated with reduced in egg production, enteritis and kidneyproblem. Kidney disease, variously described as nephrosis, nephritis or uremia was first reported in the USA. In Malaysia,local nephropathogenic infectious bronchitis virus (IBY) was first noticed in the field in 1980 and a prototype MH5365/95was isolated in 1995. In field and experimental infections the disease appears initially as respiratory infection followed byviremia and nephritis. Respiratory disease is characterized clinically by tracheal rales, coughing and sneezing. Lesionsdescribed are serous, catarrhal or caseous exudates in the trachea, nasal passages, lungs congestion around the major bronchi,and cloudiness of the air sacs are also described. Microscopically the principle tracheal lesions are heterophil andmononuclear cells infiltration of the mucosa and submucosa, congestion, epithelial hyperplasia, glandular changes anddeciliation. Ultrastructural studies by scanning electron microscopy of the trachea experimentally infected bynephropathogenic IBY shown discrete areas of non-ciliated tracheal mucosa and protruded hyperplastic mucosa into tracheallumen. Pseudo-membrane presumably consisting of inflammatory cells, degenerated cells and mucus were also noticed.The objective of this study is to examined the cytopathological changes occurred in infected trachea and kidney of chickenfollowing infection by IBY strain MH 5365/95 by light microscopy, SEM and TEM analysis.Materials and MethodsThirty-seven specific pathogen-free (SPF) chickens were used at 21-day old. The local strain MH 5365/95 was used. It hadbeen passage six times in embryonated eggs and twice in chickens, before use in this experiment. Chickens were divided into 2groups (1'~30, 2n~7). Chickens in the I st group were inoculated intratracheally with 0.2ml of viral inocula containing 10 sEIDso ml of IBV-MH 5365/95 strain. The 2nd group was acting as control (C), received 0.2ml of PBS by intratracheal route.Clinical signs were recorded daily during 24-days course of post infection (p.i), One and 3-4 chickens from control andinfected groups respectively were killed at the interval of 3,6,9, 12, IS, 18,21 and 24 days p.i. Necropsy chickens wereexamined for gross lesion in trachea and kidney. Pieces of tissues were placed into different fixatives for different analysisprocedures. H & E studies: The tissues were fixed in 10% phosphate buffered formalin and processed for paraffin block usingstandard procedure before staining with hematoxylin and eosin. Transmission studies: Pieces of trachea and kidney wereplaced in cacodylate-buffered glutaraldehyde 2.5% and cut into lmm' blocks. The tissues were first pre-fixed in glutaraldehydeat 4°C for 12 hours, post-fixed for 2 hours in osmium tetroxide, and then dehydrate and embedded in Epon. Ultrathin sectionswere cut and stained with uranyl acetate followed by lead citrate. All specimens were examined with TEM (Hitachi H-7l(0)operated at 75KV. Scanning studies: Pieces of trachea were fixed with 2.5% glutaraldehyde and post-fixed in I% osmiumtetroxide for 2 hours. The samples were washed with 0.1M sodium cacodylate buffer for 3 changes in 10 minutes, dehydratedthrough a series of acetone and subjected to critical point drying for 30 minutes. The tissues were add onto stubs using double-sided tap and coated with gold palladium in a sputter coater. Sections were viewed under electron microscope (JOEL 6400SEM) at 15 kv.Results and DiscussionMicroscopy of trachea: Infectious bronchitis virus predominantly replicates in the upper respiratory tract followed by viraemicspread to various organs namely the kidney, reproductive tissue, lymphoid tissues and mtestinal tract. Local nephropathogenicMH5365/95 IBV causes respiratory distress, nephritis and enteritis. The respirazory disease is only transient which last foronly 2 weeks and then followed by severe kidney problem. The respiratory system of control chickens appeared normal. Theirtrachea epithelial cells are covered with cilia. However, early changes in the infected tracheas are observed from day 3 pi.Epithelial cells from infected trachea showed discrete. area of cilia loss and being repiaces by microvilli. Many degeneratedepithelial cells and other tissue debris are found in the tracheal lumen. In TEM studies, infected epithelial cells showed cellswelling due to increased amount of RER and swollen mitochondria. The latter were occationally seen with increased numberof cristae. Cytopathological changes observed in this study at 3 days pi, is correlated with the tracheal ciliostasis observed in215UPM Research Report 1997-2000, Vol II,Section 2-Extended Abstractsanother study on tracheal organ culture (TOC) at 3 days pi.. Microscopy of kidney: The renal epithelial cells of control chickenappeared normal. However, in the IBV-infected chicken, the kidney were enlarged and swollen. Many tubules including theproximal convoluted, distal convoluted and collccting tubule showed degenerative changes of swollen arid vacuolation of theirepithelium, Under TEM, the early changes in the infected tubular epithelial cell were represented by cell swelling and reducedelectron density of the cytoplasmic matrix. Subsequently, there is an increased in the amount of rough endoplasmic reticulum(RER), together with the increased of fine electron-dense material in its dilated cisternae. This organelle has a highersucceptibility to infection by IBV. Free ribosomes and polyribosomes were also increased due to degranulation of severelydilated RER. The replication process of IBV in renal cells is manifested by the presence of large number of virus particles,mainly in the dilated cisternae of RER. Virus particles were found in endocytotic vesicles of the dilated cisternae of theproliferated RERConclusionsThe study provided information on structural changes in the ciliated epithelial cells of trachea and kidney during the course ofIBV infection. Being a main site for virus multiplication, cytopathological changes in the trachea are observed at 3 days pi.Cytopathological changes observed in this study at 3 days pi, is correlated with the tracheal ciliostasis observed in anotherstudy on tracheal organ culture at 3 days pi. In kidney, the virus was found abundantly in the in the dilated cisternae of RER.Virus particles were found in endocytotic vesicles of the dilated cisternae of the proliferated RER. As the chicken isrecovering from the respiratory distress, double-membrane autophagic vacuoles are found scattered in the most infectedepithelial cells indicating the activity of cellular repair.Benefits from the studyThe study provided information on cytopathological changes in the trachea and kidney of chicken following infection withlocal nephropathogenic IBV.Patent(s), if applicable:NilStage of Commercialization, if applicable:NilProject Publications in Refereed Journals1. Arshad, ss and AI-Salihi. 2003. Ultrastructure pathology of trachea in experimentally infected with Infectious bronchitis Virus MH5365/95. Annals of Microscopy.3: 43-47.2. Arshad, ss and Al-Salihi, 2003. Avian infectious Bronchitis Virus: Ultrastructural pathology of trachea and kidney of chicken infectedwith locallBV strain. Annals ofMicroscopy. 3: 48-50.Project Publications in Conference Proceedings1. Arshad, SS and Al-Salihi, 2002. Ultrastructure pathology of trachea in experimentally infected with Infectious bronchitis Virus MH5365195. InProceedingof the II th Scientific Conference 2002, Electron Microscopy SocietyMalaysia. 17-18Disember2002; p280-2842. Arshad, SS and Al-Salihi, 2002. Avian infectious BronchitisVirus: Ultrastructural pathology of trachea and kidney of chicken infected withlocallBV strain. InProceeding of the II th Scientific Conference 2002, Electron Microscopy Society Malaysia. 17-18 Disember 2002;p403-405.3. Arshad, SS and Al-Salihi. 2001. Ultrastructure Change of the Kidney of Chicken Experimentally Infectedwith Infectious Bronchitis VirusMH-5365195 Strain. In: Proceedings of the 10th Scientific Conference Electron Microscopy Society of Malaysia, Kuala Lumpur, 8_10111November2001; p270-2724. Arshad, SS and AI-Salihi.200 I. Avian InfectiousBronchitisVirusUltrastructure Pathologyof Trachea and Kidney of Chicken Infected withLocaIIBV. In: Proceeding of the Persidangan Kebangsaan Penyelidikan dan Pembangunan (P&P) Institut PengajianTinggi Awam, PWTC,Kuala Lumpur, 25-26 Oktober 200 I.5. Arshad, SS and Yap, ML. 2000. The ultrastructure oflocal isolates of avian infectious bronchitis virus. In: Proceedingof the 9"' ScientificConference ofEMSM 2000, p 226-227.Graduate ResearchNameGraduateof Research Topic Field of Expertise Degree Awarded Graduation Year216Agricultural SCiencesKarima AI-Salihi and AnimalbacteriologyPost-doctoralTransmissionScanningMicroscopychickeninfectedinfectiousbronchitis virus.tissueswithofIRPA Project numberOI-02-04-0305UPM Research Cluster:BAB217

Microscopy analysis of trachea and kidney of chicken infected with nephropathogenic infectious bronchitis virus MH5365/95

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Microscopy analysis of trachea and kidney of chicken infected with nephropathogenic infectious bronchitis virus MH5365/95

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