“STUDIES ON VISCERAL GOUT IN BROILER CHICKS WITH SPECIAL REFERENCE TO ASTROVIRUS” BY CHAUDHARI ASHISH D. B.V.Sc. & A.H. (Reg. No. 04-2444-2014) DEPARTMENT OF VETERINARY PATHOLOGY COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDRY ANAND AGRICULTURAL UNIVERSITY ANAND – 388001 (GUJARAT) 2016 “STUDIES ON VISCERAL GOUT IN BROILER CHICKS WITH SPECIAL REFERENCE TO ASTROVIRUS” A THESIS SUBMITTED TO THE ANAND AGRICULTURAL UNIVERSITY IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF Master of Veterinary Science IN VETERINARY PATHOLOGY BY CHAUDHARI ASHISH D. B.V.Sc. & A.H. (Reg. No. 04-2444-2014) DEPARTMENT OF VETERINARY PATHOLOGY COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDRY ANAND AGRICULTURAL UNIVERSITY ANAND – 388001 (GUJARAT) 2016 ABSTRACT “STUDIES ON VISCERAL GOUT IN BROILER CHICKS WITH SPECIAL REFERENCE TO ASTROVIRUS” Name of the Student Name of Major Advisor Chaudhari Ashish D. Dr. C.J. Dave Department of Veterinary Pathology College of Veterinary Science and Animal Husbandry Anand Agricultural University, Anand-388 001 Gujarat, India. Visceral gout is a syndrome of major concern causing heavy economic losses in broiler chicks and poses a challenge to control because of its multifactorial etiology. In present study, attempts were made to confirm association of Chicken astrovirus (CAstV) and Avian nephritis virus (ANV) with visceral gout. Gout is a disorder of uric acid metabolism that allows uric acid to accumulate in the blood and tissues. The present epidemiological study was carried out to know the autopsy incidence of visceral gout from April, 2011 to March, 2016 and classified year wise and month wise to ascertain the susceptibility and relative incidence of visceral gout in commercial broiler chicks up to two weeks of age and role of Astrovirus in the causation of visceral gout. Total 25 numbers of suspected carcasses from a flock of broiler chicks up to two weeks of age grossly affected with visceral gout were subjected to detailed post-mortem examination. Gross lesions were recorded and mainly kidney samples were collected in sterile manner for virological studies. Molecular studies were carried out to confirm Astrovirus. Viral genomic RNA was extracted from kidney homogenate using QIAamp viral RNA mini kit and using oligonucleotide primer pair for both CAstV and ANV. RT-PCR was carried out by OneStep RT-PCR Kit method for diagnosis purpose. Abstract During the period of five years epidemiological study, total 581 flocks were found positive for visceral gout out of 8123 total flocks received for autopsy. Year wise incidence of the disease ranged between 4.01 to 9.37% with an overall average relative incidence of 7.15%. There was slightly more number of flocks affected during summer months than winter showing influence of atmospheric temperature. Farm wise incidence and mortality recorded during the study in 25 broiler flocks varied from 0.88 to 4.21 per cent with an average of 2.20 per cent. The mortality due to CAstV among 11 broiler flocks recorded varied from 1.92 to 4.21 per cent with an average of 2.81 per cent. The lesions were of diagnostic significance and characterized by varying degree of white chalky deposition on the serosal surfaces of visceral organs like kidneys, heart, liver, proventriculus, gizzard, intestine, lungs and joints. In addition to this, the lesions in the kidneys were marked by swelling, paleness, minute pinpoint haemorrhages and uratic deposition in the parenchyma. Ureters were seen distended and filled with urates. The histopathological lesions in kidneys showed mild to severe congestion, intertubular haemorrhages, desquamation and degeneration of tubular epithelium and cystic dilatation of tubules in addition to variable degree of deposition of urate crystals in the tubular parenchyma that was confirmed by De Galantha's stain, which appeared as black needle shaped crystals. Out of 25 samples tested, 11 (44%) samples were positive for CAstV, while all samples were negative for ANV by One-step RT-PCR kit. A positive samples of CAstV revealed most prominent hisopathological lesions of moderate, multifocal, periglomerular and interstitial mononuclear cells infiltration with interstitial nephritis in kidney sections. Along with the degeneration, necrosis and desquamation of the ii Abstract epithelial cells of the proximal convoluted tubules were also observed. Cytoplasmic eosinophilic inclusion bodies and eosinophilic cytoplasmic granules in necrotized epithelial cells with interstitial nephritis were seen as the most prominent lesions in the kidney in positive samples of CAstV. iii CERTIFICATE This is to certify that I have no objection for supplying to any scientist only one copy of any part of this thesis at a time through reprographic process, if necessary for rendering reference service in a library or documentation center. Place: Anand (Chaudhari Ashishkumar D.) Date: / /2016 Dr. C. J. DAVE (Major Advisor) Dr. C. J. Dave Assistant Professor, Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand- 388 001 CERTIFICATE This is to certify that the thesis entitled “ STUDIES ON VISCERAL GOUT IN BROILER CHICKS WITH SPECIAL REFERENCE TO ASTROVIRUS” submitted by CHAUDHARI ASHISHKUMAR D. (Reg. No. 04-2444-2014) in partial fulfillment of the requirements for the award of the degree of MASTER OF VETERINARY SCIENCE in the subject of VETERINARY PATHOLOGY of the Anand Agricultural University is record of bonafide research work carried out by him under my guidance and supervision and the thesis has not previously formed the basis for the award of any degree, diploma or other similar title. Place: Anand Dr. C. J. Dave Date: / /2016 (Major Advisor) Contents CHAPTER TITLE PAGE NO. NO. I INTRODUCTION 1-5 II REVIEW OF LITERATURE 6-30 III MATERIALS AND METHODS 31-41 IV RESULTS AND DISCUSSION 42-73 V SUMMARY AND CONCLUSIONS 74-78 _ REFERENCES i-ix LIST OF TABLES Table Title Page No. No. 3.1 List of chemicals/reagents used in the study. 33 3.2 Details of Primers used for RT-PCR for CAstV. 33 3.3 Details of Primers used for RT-PCR for ANV. 34 3.4 Details of AstV field samples used in the present study. 34 -35 3.5 Composition of one-step RT-PCR reaction Mix. 40 3.6 Steps and Cycling conditions for one-step RT-PCR for CAstV 41 3.7 Steps and Cycling conditions for one-step RT-PCR for ANV 41 Year wise and month wise autopsy incidence of visceral gout 4.1 during April, 2011 to March, 2016 up to two weeks of age in 68 Broiler chicks. Age wise autopsy incidence of visceral gout during April, 2011 4.2 to March, 2016 up to two week of age in broiler chicks 69 Farm wise mortality due to Visceral gout. 4.3 70 4.4 Results of Detection of Astrovirus by RT-PCR 71 LIST OF CHARTS Title Page No. 1 Year wise incidence of visceral gout based on analysis of autopsy 72 data of the department (April, 2011 to March 2016). Month wise incidence of visceral gout based on analysis 2 72 autopsy data from April, 2011 to March 2016. Age wise per cent incidence of visceral gout based on autopsy 3 73 examination of 25 flocks under study. Season wise incidence of visceral gout based on autopsy 4 73 examination of 25 flocks under study.