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Diagnosis and Molecular Epidemiology of Bovine Rotavirus and Coronavirus in Brazil

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Diagnosis and Molecular Epidemiology of Bovine Rotavirus and Coronavirus in Brazil Sveriges lantbruksuniversitet Swedish university of Agricultural Sciences Faculty of Veterinary Medicine and Animal Science Department of Biomedical Science and Veterinary Public Health Diagnosis and Molecular Epidemiology of Bovine Rotavirus and Coronavirus in Brazil Samuel Jakobsson Uppsala 2013 Degree project within the Veterinary Medicine Program ISSN 1652-8697 Examensarbete 2013:22 SLU Sveriges lantbruksuniversitet Diagnostik och Molekylär Epidemiologi av Bovint Rotavirus och Coronavirus i Brasilien Samuel Jakobsson Handledare: Mikael Berg, Institutionen för Biomedicin och Veterinär Folkhälsovetenskap Examinator: Susanna Sternberg Lewerin, Institutionen för Biomedicin och Veterinär Folkhälsovetenskap Examensarbete inom veterinärprogrammet, Uppsala 2013 Fakulteten för veterinärmedicin och husdjursvetenskap Institutionen för Biomedicin och Veterinär Folkhälsovetenskap Kurskod: EX0751, Nivå A2E, 30hp Key words: molecular epidemiology, rotavirus, coronavirus, calf, diarrhea, Brazil Nyckelord: molekylär epidemiologi, rotavirus, coronavirus, kalvar, diarré, Brasilien Online publication of this work: http://epsilon.slu.se ISSN 1652-8697 Examensarbete 2013:22 Abstract ........................................................................................................................................... 1 Sammanfattning .............................................................................................................................. 1 Introduction ..................................................................................................................................... 2 Scope of the study ........................................................................................................................... 2 Litterature study .............................................................................................................................. 3 Brazil’s agricultural sector .................................................................................................................... 3 Neonatal diarrhea in calves .................................................................................................................. 3 The disease ....................................................................................................................................... 3 Economic impact .............................................................................................................................. 4 Management of diarrheic calves ...................................................................................................... 4 Prevention and disease control ........................................................................................................ 4 Diagnosis ........................................................................................................................................... 5 Rotavirus ................................................................................................................................................... 5 Taxonomy ............................................................................................................................................. 5 Pathogenesis ......................................................................................................................................... 6 Host spectrum ...................................................................................................................................... 6 Morphology .......................................................................................................................................... 7 Genetic structure .................................................................................................................................. 7 Replication ............................................................................................................................................ 8 Prevalence in Brazil and other parts of the world ................................................................................ 8 Molecular epidemiology ....................................................................................................................... 9 G-genotypes ..................................................................................................................................... 9 P-genotypes ...................................................................................................................................... 9 Combinations .................................................................................................................................... 9 Difference between dairy and beef calves ..................................................................................... 10 Mixed genotypes ............................................................................................................................ 10 Bovine Coronavirus ................................................................................................................................. 10 Taxonomy ........................................................................................................................................... 10 Pathogenesis ....................................................................................................................................... 11 Host spectrum .................................................................................................................................... 11 Morphology ........................................................................................................................................ 12 Genetic structure ................................................................................................................................ 12 Replication .......................................................................................................................................... 12 Prevalence in Brazil and other parts of the world .............................................................................. 14 Molecular epidemiology ..................................................................................................................... 14 Materials and methods .................................................................................................................. 15 Lab description and equipment: ......................................................................................................... 15 Sample collection and preparation .................................................................................................... 15 RNA extraction .................................................................................................................................... 15 Reverse Transcription ......................................................................................................................... 16 Multiplex semi-nested Reverse Transcription Polymerase Chain Reaction ....................................... 16 VP4 ...................................................................................................................................................... 17 VP7 ...................................................................................................................................................... 17 NSP4 .................................................................................................................................................... 18 S-gene ................................................................................................................................................. 18 Amplicon purification and sequencing ............................................................................................... 18 Sequence editing and building of phylogenetic trees ........................................................................ 19 Results ........................................................................................................................................... 20 MSN RT-PCR ........................................................................................................................................ 20 Rotavirus batch one ............................................................................................................................ 20 Rotavirus batch two ............................................................................................................................ 20 Coronavirus batch one ....................................................................................................................... 20 Coronavirus batch two ....................................................................................................................... 21 Sequencing results .................................................................................................................................. 21 Batch one ............................................................................................................................................ 21 Batch two ............................................................................................................................................ 21 Phylogenetic trees .................................................................................................................................. 22 Discussion ....................................................................................................................................
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