Historical Evolution of Animal Biotechnology 15

T A B L E O F C O N T E N T S

0. Executive Summary...... 13

1. Introduction to Animal Biotechnology...... 15 Introduction...... 15 Historical evolution of animal biotechnology...... 15 Basics of biotechnology...... 16 DNA...... 16 RNA...... 16 Genes...... 17 Single nucleotide polymorphisms...... 17 Copy number variations in the genome...... 17 DNA sequences...... 18 Gene expression...... 18 Gene regulation...... 19 Proteins...... 19 Functions of proteins...... 19 Recombinant proteins...... 20 Monoclonal antibodies...... 21 Animal genetics...... 21 Molecular genetics...... 21 Twinning in cattle...... 22 Pig genetics...... 22 Genetic studies in dogs...... 22 Animal genomics...... 22 The mouse genome...... 22 The dog genome...... 23 Sequencing of the dog genome...... 23 Comparison of genomes of healthy and diseased dogs...... 25 Analysis of DNA copy number variation...... 25 The cat genome...... 26 Marsupial genomes...... 26 Genomes of non-human primates...... 26 Chimpanzee genome...... 26 Genome of the rhesus macaque...... 27 Livestock genomics...... 27 Bovine genome...... 28 Bovine SNP map...... 29 Pig genome...... 29 Horse genome...... 30 Sheep genome...... 31 Chicken genome...... 31 Turkey genome...... 32 Salmon genome...... 33 Priority genome list of the National Human Genome Research Institute...... 33 Applications of animal genomics...... 34 Genomics of disease resistance...... 35 Statistical genomics to improve breeding...... 35 Chicken breeding based on genomics...... 35 Animal proteomics...... 36 Applications of proteomics in animals...... 36 Caseins in goat milk...... 36 Lactic acid bacteria...... 37 Applications of proteomics in animal healthcare...... 37 Bioinformatics...... 37 Application of nanobiotechnology for animal health...... 38 Biomarkers and animal health...... 39 Recombinant protein manufacture...... 39 Animal biotechnology in relation to other technologies...... 39

2. Application of Biotechnology in Animals...... 41 Introduction...... 41 Genetic engineering...... 41 Livestock improvement by genetic engineering...... 41 Disease control by genetic engineering...... 41 Limitations and precautions for genetic engineering...... 42 Transgenic animal technology...... 42 Cloning animals...... 43 Nuclear transfer technology...... 43 Nuclear bisection for cloning...... 45 Zona-free cloning method...... 45 Abnormalities in cloned animals...... 46 Cloning from embyonic cells...... 47 Cloning of rabbits...... 47 Cloning the rat...... 48 Cloning the horse...... 48 Cloning the cow...... 48 Cloning the dog...... 49 Cloning in primates...... 49 Retrovector-mediated production of transgenic animals...... 49 Episomal vector-mediated gene delivery...... 50 Sperm-mediated gene transfer...... 50 Lentiviral transduction of male germ-line stem cells...... 51 Lentiviral transgenesis...... 52 Transgenic pharmaceuticals...... 52 Proteins from the milk of transgenic animals...... 52 Advantages of milk as source of transgenic proteins...... 53 Therapeutic proteins from rabbit milk...... 54 Recombinant human antibodies from cows...... 55 Therapeutic proteins from goat milk...... 55 Chicken transgenesis for the production of biopharmaceuticals...... 56 Concluding remarks about production of recombinant proteins in animals...... 56 Companies involved in production of transgenic pharmaceuticals...... 56 Transgenic food products...... 57 Milking genetically modified cows...... 57 Transgenic fish...... 57 Cloned animals as sources of milk and meat...... 58 Animal feeds from transgenic plants...... 58 Transgenic modification of plants to increase nutritional value of animal feeds. .59 Transgenic disease models...... 59 Technologies to create transgenic disease models...... 59 Gene manipulation techniques...... 59 Embryonic stem cells for gene targeting...... 60 Homologous recombination...... 60 Animal models of human diseases...... 61 Transgenic models for studying human drug metabolism and toxicity...... 61 The Human Genome Project and the role of transgenics...... 62 Genomic and proteomic analyses of transgenic animal models...... 62 Concern about health and welfare of transgenic animals...... 63 Safety of transgenic technology...... 63 Concluding remarks about use of transgenic animals...... 64 RNA interference technology...... 64 RNAi versus antisense...... 64 Applications of RNAi in animal biotechnology...... 64 Xenotransplantation...... 65 Pigs for xenotransplantation...... 65 Genetically engineered pigs for transplants...... 66 Risks of xenotransplantation...... 66 World Health Organization and xenotransplantation...... 67 Ethical aspects of animal biotechnology...... 67

3. A Biotechnology Perspective of Animals Diseases...... 69 Introduction...... 69 Infections in animals...... 69 Viral infections...... 70 Avian influenza...... 70 Animal surveillance of influenza...... 72 Animal biotechnology implications of H1N1 influenza...... 73 Animal corona viruses and human SARS...... 73 Avian coronavirus...... 74 Bluetongue virus...... 75 Canine parvovirus...... 75 Classical swine fever...... 75 Developing new treatments against FMD...... 75 Equine infectious anemia...... 76 Foot-and-mouth disease...... 77 Porcine reproductive and respiratory syndrome virus...... 78 Rabies...... 79 Rinderpest...... 79 Bacterial infections...... 79 Bovine tuberculosis...... 79 Mycoplasmal pneumonia...... 80 Protozoal infections...... 80 Coccidiosis...... 80 Neosporosis...... 81 Toxoplasmosis...... 81 Trypanosomiasis...... 81 Nematodes...... 82 Infections that cross the species barrier...... 82 Complications of bacterial infections and antibiotic use in animals...... 82 Transmissible spongiform encephalopathies (TSEs)...... 83 Inter-species transfer of prions...... 83 Scrapie...... 83 Bovine spongiform encephalopathy...... 84 Epidemiology of BSE...... 84 Biomarkers in the urine of BSE infected cattle...... 85 Human health implications of BSE...... 85 Breeding animals protected against BSE...... 86 TSE research...... 86 Prion gene haplotyping...... 86 Pharmacological approaches to TSE research...... 86 Molecular diagnostic approach to TSE research...... 87 RNAi for knockdown of the bovine prion gene...... 87 Chronic wasting disease...... 88 Chronic wasting disease in wildlife...... 88 Chronic wasting disease of the cattle in Sudan...... 89 Chronic wasting in dairy cows in the Netherlands...... 89 Genetic disorders in farm animals...... 89 Genetic predisposition to acquired diseases in animals...... 90 Diseases of pet animals...... 90 Canine anemia...... 90 Cardiovascular disease...... 90 Heart failure...... 91 Cardiac complications of canine babesiosis...... 91 Diabetes...... 91 Role of biotechnology in management of diabetes...... 92 Arthritis...... 92 Cancer in cats and dogs...... 93 Cancer clinical trials in dogs...... 93 Canine Comparative Oncology Genomics Consortium...... 94 Preventive veterinary medicine...... 94 Prevention of introduction of foreign animal diseases...... 94 Producing transgenic cattle resistant to BSE...... 95 Zoonotic diseases...... 95 Collaborative management of animal and human health...... 96 Vaccines for zoonotic viral diseases...... 96

4. Molecular Diagnostics in Animals...... 97 Introduction...... 97 Nucleic acid technologies...... 97 The polymerase chain reaction...... 97 Basic Principles of PCR...... 97 Target selection...... 98 Detection of amplified DNA...... 98 Real-time PCR systems...... 98 LightCycler PCR system...... 99 Molecular beacons...... 99 Applications of PCR in veterinary medicine...... 99 Fluorescent in situ hybridization...... 100 Immunodiagnostics...... 102 Enzyme-linked immunoassays...... 102 Bovine Gamma Interferon Test...... 102 Antigen diagnosis of trichinosis...... 103 Parachek™ for the diagnosis of Johne's disease...... 103 Antibodies for differentiation between vaccinated and infected animals...... 104 Biochip/microarray technology...... 104 Applications of microarrays in animal biotechnology...... 105 Cattlearray3800 for functional genomics...... 105 eSensor™ electrochemical biochip...... 106 FR 48 microfluidic biochip...... 106 Biosensors...... 106 Immunosensors...... 107 Biosensor for ovulation prediction in dairy cows...... 107 Flow cytometry for animal diagnostics...... 108 Molecular imaging in animals...... 108 Veterinary cytogenetics...... 109 Applications of molecular diagnostics in animals...... 109 Canine DNA testing...... 110 Diagnostic aids to selective breeding...... 110 Selection of desirable traits...... 110 Using genetic markers for improved milk production in dairy cattle...... 111 Application of bovine genomics for improving milk yield...... 111 Recognition of hereditary syndromes...... 112 Genetic markers in animals...... 112 SNP genotyping in animals...... 112 SNP genotyping for selective breeding of chicken...... 112 Animal identity and parentage analysis...... 113 Animal species identification in food...... 113 Diagnosis of infections...... 114 Bacterial infections...... 114 Diagnosis of viral infections...... 114 Molecular diagnosis of avian influenza...... 116 Diagnosis of parasitic infections...... 117 Detection of natural or bioterror threats to livestock...... 118 Molecular diagnosis of prion diseases...... 118 Bovine spongiform encephalopathy...... 118 Testing for BSE in living animals...... 120 Prions in urine...... 120 Diagnosis of chronic wasting disease in wildlife...... 121 Developing new tests for prion diseases...... 121 Differentiation among various types of TSEs...... 121 Protein cyclic amplification...... 122 Antibody tests for prion diseases...... 122 Scrapie genotyping...... 122 A real-time ultrasonic method for prion protein detection...... 123 Companies involved in developing molecular diagnostics for TSEs...... 123 Diagnosis of genetic disorders...... 124 Genetic screening of companion animals...... 124 Genes associated with exercise-induced collapse...... 124 Preimplantation genetic diagnosis...... 124 Diagnosis of cancer in animals...... 125 Diagnosis of food-borne pathogens...... 125 Introduction...... 125 Molecular diagnostic methods used in food-borne infections...... 126 Limitations of use of molecular probes in food analysis...... 126 Companies with technologies for food pathogen detection...... 127 Biotechnology-based novel diagnostics for aquatic animals...... 128 Detection of chemicals in foods of animal origin...... 128 Companies developing molecular diagnostics for animals...... 129

5. Biotechnology-based Veterinary Medicine...... 131 Introduction...... 131 Biotechnology versus pharmaceutical products...... 131 Role of biotechnology in drug discovery and development...... 132 Cost of veterinary vs. human drug discovery and development...... 132 Advantages and disadvantages of testing biotech products in animal models. .133 Biotechnolgoy-based antiparasitic drugs...... 133 Non-antibiotic strategies for control of infections in animals...... 133 Probiotics...... 134 Potential role for probiotics in the human gut...... 134 Potential role for probiotics in animals...... 134 Probiotic bacteria for control of pathogens in cattle...... 134 Nonantibiotic drugs for infections in animals...... 135 Immunomodulation as an alternative to antibiotics in infections...... 136 Cathelicidins: effector molecules of mammalian innate immunity...... 136 Bacteriophage therapy for antibiotic resistance...... 136 Biotechnology for treating tendon injuries...... 137 Use of growth factors to facilitate tendon injuries...... 137 Productivity enhancers...... 137 Bovine somatotropin for increasing milk production in dairy cows...... 138 Increasing milk production in cows by feeding propionibacteria...... 139 Use of growth factors...... 139 Transgenic plant products for use in animals...... 139 Biotechnology-based vaccines...... 140 Modern vaccines without viral non-structural proteins...... 140 Plant-derived vaccines for use in animals...... 141 Nano-bead vaccine adjuvant...... 142 Genetically engineered vaccines...... 142 Application of nucleic acid vaccines in veterinary medicine...... 142 DNA vaccines...... 142 DNA vaccine for tuberculosis...... 144 DNA vaccines for West Nile encephalitis...... 144 Gene-based vaccine for Marek’s disease...... 145 Genetic engineering of live rabies vaccines...... 146 Genetically engineered vaccines for equine encephalitis...... 146 Genetically engineered vaccines for Johne's disease...... 146 Vaccines against avian influenza...... 147 Vaccines against parasitic infections...... 148 Recombinant marker vaccines...... 148 Marker vaccines for foot-and-mouth disease...... 148 Marker vaccine for Newcastle disease...... 149 Vaccines for classical swine fever...... 149 Vaccines for tick control...... 150 Vaccination to protection swine from H1N1 influenza virus infection...... 150 Vaccination of cattle to prevent E. coli transmission to consumers in meat...... 150 Vaccines for bacterial equine respiratory infections...... 151 Using RNAi to develop vaccines for viral infections in prawns...... 151 Companies developing biotechnology-based vaccines...... 152 Biotechnology in treatment of parasitic infections...... 152 Biotechnology in the treatment of CNS injuries in pet animals...... 153 Paraplegia due to acute spinal cord injury in dogs...... 153 RNAi for suppression prions in livestock...... 153 Cell Therapy...... 154 Umbilical cord blood stem cells...... 154 Application of stem cells in veterinary medicine...... 154 Use of stem cells to repair tendon injuries in horses...... 154 Stem cells for spinal cord injury in dogs...... 155 Gene therapy...... 155 Gene therapy vectors...... 156 Gene therapy by mitochondrial transfer...... 156 In utero gene therapy...... 157 Applications of gene therapy in veterinary medicine...... 157 Gene therapy for mucopolysaccharidosis VII in dogs...... 157 Gene therapy to increase disease resistance...... 158 Gene therapy for infections...... 158 Gene therapy for hematological disorders...... 158 Gene therapy for cardiomyopathy in dogs...... 159 Gene therapy for endocrine disorders...... 159 Gene therapy for arthritis...... 160 Gene therapy for renal failure...... 160 Cancer gene therapy...... 160 Antiangiogeneic cancer gene therapy in dogs...... 161 Brain tumors in cats and dogs...... 161 Breast cancer in dogs...... 162 Canine hemangiosarcoma...... 162 Canine melanoma...... 163 Canine soft tissue sarcoma...... 164 Melanoma in horses...... 164

6. Research in Animal Biotechnology...... 165 Introduction...... 165 Research institutes...... 165 Animal and Natural Resources Institute (USDA)...... 165 Center for Animal Biotechnology at University of Melbourne (Australia)...... 166 CSIRO Livestock Industries...... 167 Danish Veterinary Institute...... 168 Friedrich-Loeffler-Institute...... 168 Indian Veterinary Research Institute...... 169 Institute for Animal Health of UK...... 169 Kimron Veterinary Institute...... 170 Korean National Livestock Research Institute...... 170 National Agricultural & Veterinary Biotechnology Center of Ireland...... 171 Swiss Federal Institute of Technology...... 171 Veterinary Laboratories Agency of UK...... 172 Veterinary Medical University of Vienna...... 173 Ethical issues of research in animal biotechnology...... 173 Future prospects...... 173 Strategies for control of twining in cattle...... 174 Future developments of molecular diagnostics...... 174 Future of vaccine application in veterinary medicine...... 174 Promotion of innate immunity in animals...... 175 Identification of key parasite antigens for eliciting immune response...... 175 Virus-like particle vaccines for lasting immune response...... 175 Control of respiratory virus infections...... 176 Control and prevention of bioterrorism diseases in animals...... 176 Genetic control of disease resistance...... 176 Production of cattle lacking prion protein...... 177 Application of genetics and biotechnology to wildlife management...... 177 Future of animal genomics...... 178 Future prospects of in vitro meat production...... 178

7. Animal Biotechnology Markets...... 181 Introduction...... 181 Markets for biotechnology-based products for animal healthcare...... 182 Markets for biopharmaceuticals for animals...... 183 Markets for recombinant proteins for animal healthcare...... 183 Markets for vaccines for animals...... 184 Markets for animal diagnostics...... 184 Test for bovine spongiform encephalopathy...... 184 Animal biotechnology markets according to therapeutic areas...... 184 Markets for biotechnology-based animal products for humans...... 185 Transgenic proteins...... 186 Market for xenotransplantation...... 186 Strategies for promoting use of animal biotechnology...... 187 Financial losses from death and disease in animals...... 187 Losses in farm animals...... 187 Losses in poultry...... 187 Losses in equine industry...... 187 The emerging role of pet owners...... 187 Improvement in cattle through application of biotechnology...... 188 Economic aspects of genomic evaluation of dairy cattle...... 188 Pig market...... 188 Cattle Market...... 189 Poultry market...... 189 Milk from genetically modified cows...... 189 Transgenic fish...... 190 Role of biotechnology in livestock performace enhancer market...... 190 Gene transfer technologies...... 190 In vitro meat production and animal biotechnology markets...... 190 Cost-benefit aspects of transgenic proteins...... 190 Lower costs of transgenic production...... 190 Lower costs of treatment...... 191 Unmet needs in animal biotechnology...... 191 Future opportunities for biotechnology in animal healthcare...... 192 Farm animals...... 192 Companion animals...... 193

8. Regulatory issues...... 195 Introduction...... 195 Regulatory agencies for veterinary biotechnology in the US...... 195 FDA regulatory issues in agricultural biotechnology...... 196 FDA guidlines on use of antibiotics in food-producing animals...... 197 Food safety evaluation of transgenic animals...... 198 Food from cloned animals...... 199 FDA investigation of drug transfer into eggs...... 200 Animal feed safety...... 201 Medicated feeds...... 201 Regulatory issues for production of transgenic proteins...... 202 Risks of animal biotechnology...... 202 FDA regulation of bovine products...... 203 Worldwide biotechnology regulatory and trade issues...... 203 9. Companies Involved in Animal Biotechnology...... 205 Introduction...... 205 Biotechnology at top veterinary pharmaceutical companies...... 205 Profiles of selected companies...... 205 Collaborations...... 319

10. References...... 323

Tables

Table 1-1: Landmarks in the evolution of animal biotechnology in the 20th century.....15 Table 1-2: Expression systems for production of recombinant proteins...... 20 Table 1-3: Applications of genomics in livestock industry and veterinary medicine...... 34 Table 1-4: Applications of proteomics in livestock industry and veterinary medicine....36 Table 1-5: Selected animal genomics and proteomics databases (DB)...... 38 Table 2-1: Recombinant proteins obtained from milk of transgenic animals...... 54 Table 2-2: Companies involved in the production of transgenic pharmaceuticals...... 57 Table 2-3: A comparison of gene knockout and transgenic techniques...... 60 Table 2-4: Examples of transgenic mouse models of non-neoplastic human diseases...61 Table 3-1: Diseases of dairy cattle...... 69 Table 3-2: Causes of chronic wasting disease in animals...... 88 Table 4-1: Potential applications of microarrays in animal biotechnology...... 105 Table 4-2: Biosensor technologies with potential applications in molecular diagnostics ...... 107 Table 4-3: Applications of molecular diagnostics in animals...... 109 Table 4-4: Viruses that can be detected by molecular diagnostics...... 114 Table 4-5: Testing for harmful prions in brain tissue from dead cattle...... 119 Table 4-6: Companies involved in developing molecular diagnostics for TSEs...... 123 Table 4-7: Pathogenic bacteria in food and targets for molecular diagnostic probes. . .126 Table 4-8: Companies involved in molecular diagnostics for food-borne infections.....127 Table 4-9: Companies developing molecular diagnostics for veterinary medicine...... 129 Table 5-1: Veterinary biotechnology products...... 131 Table 5-2: Pharmaceutical versus biotechnology products...... 132 Table 5-3: Nonantibiotic strategies for control of infections...... 133 Table 5-4: Experimental DNA vaccines tested in animals...... 143 Table 5-5: Companies developing biotechnology-based vaccines for animals...... 152 Table 6-1: Areas for future research applications of animal biotechnologies...... 174 Table 7-1: Worldwide markets for biotechnology-based products for farm animals: 2009-2019...... 182 Table 7-2: Worldwide markets for biotechnology-based products for pet animals: 2009- 2019...... 182 Table 7-3: Biotechnology-based markets for animal healthcare according to regions: 2009-2019...... 183 Table 7-4: Biotechnology markets for farm animals according to therapeutic areas: 2009-2019...... 185 Table 7-5: Biotechnology markets for pet animals in therapeutic areas: 2009-2019...185 Table 7-6: Worldwide markets for biotechnology-based animal products for humans: 2009-2019...... 186 Table 9-1: Ranking of top 5 veterinary companies with biotechnology products...... 205 Table 9-2: Selected collaborations of companies in animal biotechnology...... 319

Figures

Figure 1-1: Relation of animal biotechnology to other technologies and human health. 40 Figure 2-1: Nuclear transfer technology...... 44 Figure 2-2: Generation of transgenic animals by linker based sperm-mediated gene transfer...... 51 Figure 2-3: Production of therapeutic proteins in the milk of transgenic animals...... 53 Figure 7-1: Unmet needs in animal biotechnology...... 192