Advantages

Advantages of Domestic Species as Biomedical Models

Goals

a) to present examples of recent and historical milestones in the uses of domestic species for biomedical research, and b) to provide examples of domestic species as biomedical models that may be under-funded or not funded by NIH.

Since 1901 there have been 17 Nobel Prizes awarded to scientists that used cows, chickens, swine, horse and sheep as biomedical models during their studies [1]. General research areas that potentially could be advanced by use of these domestic species as biomedical models are listed in Table 1. The following are specific examples of some of the advantages and uses of domestic species as biomedical models:

 

Examples of Historical and Recent Milestones in Use of Domestic Species for Biomedical Research

 

Immunology and Immunobiology:

Vaccination: Edward Jenner used the cowpox virus for the first vaccine against small pox [2], which paved the way for modern vaccination programs against smallpox and other human and animal plagues [3].

Acquired Immunological Tolerance: John Hunter first described freemartin cattle (female born co-twin with a male) [4], and Lillie dissected placenta of freemartins and first described the intermingling of blood between twins [5]. By 1951, Medawar (shared the Nobel Prize in Physiology in 1960 with Burnet) reported successful skin allografts between freemartins and normal males [6]. Macfarlane Burnet discovered that vascular anastomosis results in blood chimerism in dizygotic twins in cattle and this explains the tolerance to allografts between twins. This work in cattle formed the basis for Dr. Burnet’s Nobel Prize in Physiology in 1960 [7]. It also explained in part the freemartin condition in female calves born co-twin with a male and has been followed by a series of studies on microchimerism in parous women [8].

Humoral Immunity: Glick and colleagues reported that the avian bursa of Fabricius was necessary for humoral immunity and antibody production [9], which was later followed by identification of B lymphocytes and acceptance of the idea that lymphocytes are responsible for adaptive immunity [10].

 

Xenotransplantation:

Since 1960, hundreds of thousands of heart valves of pigs and cattle have been implanted into human hearts (24,000 annually in U.S. and 68,000 worldwide) [11].

Cadaveric or live organ transplants do not meet the demand for organs. Although porcine xenografts may be the solution to this problem [3], the surface of pig cells contain α-1,3-galactosyltransferase, and primates including humans contain preformed antibodies against epitopes on this molecule. Consequently, use of pig organs for transplantation into humans is severely hampered. Recently, nuclear transfer technology produced α-1,3-galactosyltransferase “knockout” pigs. Hearts and kidneys from these pigs have been successfully transplanted without hyperacute rejection and severe microangiopathy into primates [12-15]. These major scientific breakthroughs are expected to greatly enhance the potential use of pigs as a source of donor organs, tissues and cells for humans [16-19].

Porcine islets can restore euglycemia in animal models thus pigs may also be a source for islets to treat diabetes in humans [20].


Reproductive biology:

Artificial Insemination was initially practiced in horses in 1322 then more extensively in bitches and horses in the late 1700’s and 1800’s. By the 1930’s, AI was used commercially in 1000’s of mares, cows and sheep [21, 22].

Cryopreservation of sperm was accomplished first in cattle [23] and later applied to humans [24].

Superovulation and embryo transfer: Although Heape performed the first embryo transfer in rabbits in the 1890’s, the revival of interest, initial transfers in species other than rabbits, and potential application of superovulation and embryo transfer to improve reproductive efficiency and enhance genetic selection were pioneered beginning in the 1930’s by Warwick, Berry and Horlacher and later by Hammond, Rowson and Willett and others in goats, sheep and cattle [25].

Cloning & nuclear transfer: Ian Wilmut used nuclear transfer procedures to successfully clone the first mammal, a sheep named Dolly, from a non-reproductive adult cell [26].

 

Developmental Biology:

Because of the ease of manipulating developing limbs in vivo, chickens have provided fate maps and enabled scientists to determine the cell-cell interactions that result in limb pattern. Indeed, over the past 15 years, the role of an increasing number of developmentally important genes has been uncovered. The principles that underlie limb development in chickens are applicable to other vertebrates including humans and there are increasing linkages with clinical genetics [27].


Cardiovascular Disease:

Heart failure occurs in at least 400,000 people annually in the U.S. alone [28]. While genetically engineered mice are excellent models [28], large animals such as calves and pigs, which have a heart size, musculature, geometry and physiology similar to humans [29], are more appropriate models for surgery, hemodynamics [30], artificial heart development [29-31], and etiology of heart failure [28].

 

Biochemistry:

Isolation and characterization of thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH): Pigs and sheep were used as sources of millions of hypothalami for the isolation and peptide sequence determination of TRH and GnRH by Schally and Guillemin, which led to the Nobel Prize for both scientists in 1977 [32, 33].

Prostaglandins were isolated from sheep seminal vesicles, crystallized, and structures of PGE and PGF determined in a series of studies by Bergstrom and Sjovall from 1957 to 1960 [34]. Bergstrom was awarded the Nobel Prize in 1982 along with Samuelsson and Vane for their work on prostaglandins [34].


Nutrition:

Milk fat secretion studies in ruminants led to the first models for understanding lipid synthesis and nutrient utilization. [35, 36]. This work conducted in the 1950’s and 60’s provided some of the seminal data on nutrient content in milk for nourishment of the neonate. In addition, the ultrastructural characterization of the mammary gland and resulting micrographs continue to be used to provide a basic understanding of milk secretion in humans.

 

Metabolic Diseases:

Malignant hyperthermia is a potentially deadly disorder triggered by anesthetic agents and resulting in uncontrolled skeletal muscle calcium flux and organ failure. The stress-susceptible pig model provided important clues to the genes involved in this disorder in humans and continues to be the primary animal model to elucidate the physiological and genetic basis for this disorder (for review see: [37]).

Diabetes: the source of insulin for treatment of diabetes in humans was primarily of bovine and porcine origin from 1920 to 1982 [38].

Osteoporosis: Early work in the chicken led to the understanding of the importance of calcium metabolism and skeletal integrity [39] as well as the central role played by vitamin D in calcium absorption [40, 41].


Neurodegenerative Diseases:

Prions were first isolated by Prusiner (awarded Nobel Prize in Physiology or Medicine in 1997) from sheep and goats [42] and determined to be proteinaceous infectious pathogens that cause fatal neurodegenerative diseases including bovine spongiform encephalopathy (BSE), scrapie in sheep, and Creutzfeldt-Jakob disease (CJD) in humans [43].


New Therapeutics including pharming:

Interferon-τ, a novel cytokine discovered in sheep [44], is in clinical trials to evaluate its effectiveness as a treatment for multiple sclerosis [45-48].

Uteroferrin or TRAP (tartrate resistant acid phosphatase), a purple iron-binding glycoprotein with acid phosphatase activity, was isolated from uterine secretions of pigs [49]. This protein is a hematopoietic growth factor used to diagnose hairy cell leukemia, bone metastases in breast cancer patients [50] and bone turnover during osteoporosos [51, 52]. Pending approval TRAP may be used as a coating to improve bone grafts, bone cements and prosthetic devices by recruiting osteoclast progenitor cells from bone marrow or blood [53].

Inhibin-A and activin-A were first isolated from follicular fluids of sheep, cattle and pigs [54-58]. Currently, these dimeric glycoproteins are used as markers to diagnose Down Syndrome, and they may have value to diagnose complications associated with problem pregnancies in women including pre-eclampsia, early detection of pregnancy post IVF, miscarriages, and preterm labor, as well as ovarian cancer [59].

AMP or cationic anti-microbial peptides such as Iseganan and Micrologix are new anti-microbial agents derived from porcine leucocytes or bovine neutrophils that are in Phase II and III clinical trials [3].

Pharming: 17 different proteins are produced in cows, goats, sheep and pigs, and 10 of these proteins are produced at commercially feasible levels (>1 g liter) [60, 61] and many are in clinical trials [3]. For example, recombinant human α1-antitrypsin is produced in sheep to treat patients with pulmonary emphysema, a crippling lung disease caused by a lack of α1-antitrypsin in blood. Bovine lactoferrin, an iron-binding glycoprotein involved in host defense against infection and excessive inflammation, inhibits hepatitis C virus viremia in patients with chronic hepatitis C [62] and may have a novel role in improving cancer therapies [63]. The crystalline structure of human recombinant lactoferrin produced by transgenic cows is nearly identical to human lactoferrin [64]. Human recombinant anti-thrombin-III produced in goats is used as an anticoagulant to prevent thrombosis during surgery or delivery [65].


Current examples of use of domestic species as biomedical models that may not be funded or may be under-funded by NIH


Diseases and disorders:

Osteoporosis: Aged ovariectomized sheep fed calcium-wasting diets are a promising model for post menopausal osteoporosis because they are docile, easy to handle and house, and their bones are large enough to evaluate orthopedic implants [66]. Moreover, their skeletal turnover kinetics, hormonal profiles such as osteocalcin, responses of osteoblasts to hormones, and responses of sheep to estrogen replacement therapies are similar to humans [66].

Diabetes-induced accelerated atherosclerosis: Some 16 million people in the US have diabetes and 56 million may be insulin resistant [67]. Individuals with diabetes or insulin resistance have a 2- to 6-fold greater risk of developing atherosclerosis [67, 68]. Indeed, the most common cause of death in adult diabetics is coronary disease [69]. Swine have a cardiovascular system, metabolism and lipoprotein profiles similar to humans, and they develop spontaneous atherosclerosis with increased aging (see references in: [70]). Thus, the diabetes-induced accelerated atherosclerosis swine model, which has been well characterized, may be useful to elucidate the mechanisms causing accelerated atherosclerotic disease in humans with diabetes [70]. Even more useful to study diabetes and heart disease may be the feral pigs of Ossabaw Island, Georgia, because they develop precursors of diabetes and heart disease including raised blood pressure and hardened arteries within months of a regimen of high-fat diet and low exercise [71].

Anaphylaxis: In the U.S. it is estimated that there are 29,000 anaphylactic reactions to foods, especially peanuts, tree nuts, fish and shellfish, treated in emergency rooms and 125 to 150 deaths each year [72]. Pigs and calves are excellent models to study the mechanisms and treatments for allergic reactions to food because the gastrointestinal physiology and development of mucosal immunity in neonatal pigs is similar to humans [73], and neonatal swine and calves have hypersensitivity responses and adverse reactions similar to human allergic disease [74].

Asthma: It is estimated that 3 to 10% of the world’s population suffer from asthma and 100,000 die annually. Although allergic asthma does not occur in animals, animal models are critical to successful development of new therapies to treat this complex disease [75]. Sheep, horses and pigs for example have many asthma symptoms similar to humans, but may be an underutilized model for this disorder [75].

Sepsis: Sepsis occurs in 700,000 people and causes about 210,000 deaths annually in the U.S. alone [76]. Use of animal models to aid in development of new therapies to combat sepsis has met with little success because of the difficulty mimicking the clinical situation in humans leading to sepsis [77]. However, domestic species models enable investigators to evaluate therapies under conditions approximating clinical practice [78]. For example, sheep forced to inhale smoke and treated with bacteria undergo hyperdynamic sepsis similar to humans [79].


Reproductive biology:

Although ~123,000 cycles of assisted reproductive technology (ART) conducted in the 399 fertility clinics in the U.S. in 2003 resulted in ~36,000 births, 70% of the ART cycles were unsuccessful [80]. The recent discovery that women exhibit multiple FSH-induced wave-like patterns of follicular development throughout menstrual cycles [81-83] implies that cattle and horses, which like humans have well characterized follicular waves [84, 85] and a functional corpus luteum during their estrous cycles, may have significant advantages over the more traditional rodent models, especially for studies related to regulation of follicular development and oocyte quality during menstrual cycles to improve the success of assisted reproductive technologies [80].

 

Nutrition:

Atherosclerosis: Because of the similarity in size and other physiological, biochemical and anatomical properties to humans, including the tendency to overeat, pigs have advantages over rodent models to study atherosclerosis and cardiovascular disease. For example, feeding high-fat cholesterol diets to pigs results in atherosclerosis-like symptoms [86], but unless mouse models are genetically modified they are resistant [87].

Food intake: The dairy cow is a unique animal model to study the complex interactions of short-term signals regulating food intake, especially because the cow’s strong drive to eat, docile nature, and large size make it ideal to simultaneously monitor digestion and adsorption kinetics, endocrine responses, gene expression, metabolite pools and fluxes, and feeding behavior in response to various treatments [88].

Nutrition and food safety: Pigs and humans have a similar GI anatomy, morphology and physiology and thus have advantages over rodents as a biomedical model for nutrition studies as well as to evaluate safety of new food additives (see references in [89]). For example, the pig is an appropriate model to test the effects of neonatal Fe deficiency on anemia and the immune system [90] and effects of olestra, a fat substitute added commercially to foods, on nutrient availability [89].


Gene therapy:

Electrogene therapy was used to transfer the gene for a protease-resistant form of growth hormone releasing hormone (GHRH) into muscles of pigs, which increases secretion of growth hormone and growth for over 65 days [91]. Because of the similar physiology and anatomy of pigs and humans, pigs may be a more useful biomedical model compared with rodents to improve gene therapy to correct defects of GHRH secretion, which characterize conditions such as Turner’s syndrome, hypochrondroplasia, Crohn’s disease, intrauterine growth retardation or renal insufficiency [3].


Alcoholism and related disorder:

Pigs are the only species other than humans that prefer alcohol. In addition, their brain growth velocity is similar to humans and they are intelligent [92]. However, pigs may be underutilized as a model not only for alcoholism [93], but also effects of alcohol on prenatal development [92, 94] and alcohol induced atrial tachyarrhythmias, e.g., “holiday heart syndrome”. [95]. Sheep also have significant advantages over rodents for studies on prenatal effects of alcohol because they have brain growth and gestation characteristics similar to humans and are well established as models for fetal physiology and long-term instrumentation [92].


Oncology:

Lung cancer kills more than one million people annually [96] and adenocarcinoma is increasing in frequency and a significant cause of lung cancers [97]. Because sheep develop ovine pulmonary adenocarcinoma (incidence is 2 to 30%, see references in [98]), a naturally occurring adenocarcinoma caused by jaagsiekte retrovirus, they are an excellent model to test therapeutic agents and to study the pathogenesis, natural history, epidemiology and host susceptibility of the disease [98].

Cutaneous melanoma is one of the most deadly forms of cancer and it is increasing at a rate faster than any other cancer [99, 100]. Some lines of minipigs have a high incidence of melanoma and develop spontaneous melanomas before 3 months of age thus they have advantages over rodent models, especially because spontaneous melanomas are rare in rodents, and precursor melanoma cells in genetic “knockout” mice are from the dermis rather than the epidermis as in humans [100, 101].

Ovarian cancer afflicts ~1 of 70 women in the US and it is one of the most lethal and difficult to detect (fewer than 25% detected at curable stage) of cancers that afflict women [102, 103]. Chickens are an important biomedical model for etiological studies and to evaluate chemopreventive and therapeutic agents because they are the only known species other than humans that exhibit spontaneous ovarian cancer resulting from transformation of ovarian surface epithelial cells [102-106].

 

Genomics:

Genomic variation is clearly a major factor in host resistance to pathogens in humans and animals. Identification of specific genome sequences that predispose susceptibility/resistance to disease will be fundamental to averting accidental or terrorist-initiated epidemics, and to developing models of human gene/pathogen interaction. Genomic variation also underlies traits such as growth, body composition, lactation, and reproductive health. The working draft of genome sequences of agriculturally important animals will, therefore, provide an invaluable resource for discovery of genes and their functions to benefit human health, animal health and production animal agriculture [107]. For example, numerous advances have been made in swine biomedical model genomics. For a review, see [108]. Moreover, insights from genomics in domestic species have already contributed to the discovery of human diseases [109]. Specifically, a mutation in the MC4R gene is associated with fatness in pigs [110] and also results in obesity in humans [111], and discovery of the limbin gene that causes chondrodysplastic dwarfism in Japanese Brown cattle [112] aided in discovery of the gene responsible for Ellis-van Creneld syndrome [113], which causes dwarfism in humans [109].

Quantitative traits: Cattle have long been studied for complex traits influenced by multiple genes as well as environmental factors. These so called “quantitative traits” are now targeted by the human health research community. Cardiovascular health, obesity, and several cancers are examples of complex traits segregating in breeding populations of cattle. The understanding of what makes cattle breeds different with respect to reproduction, lactation, growth, bone structure, fat deposition, altitude and heat tolerance, and resistance to specific pathogens will be invaluable in elucidating related physiological processes important to human health.


Infectious disease and vaccine development:

Pathogenesis and immunity are best studied in animal models in which the pathogen is naturally occurring. While there are infectious diseases in which rodents serve as relevant models, restriction to rodents, primarily mice, ignores the diversity of animal species with microbial diseases that recapitulate the human disease and represents a loss of research opportunity when the most representative model is not investigated. This often reflects a lack of knowledge in the biomedical community of the occurrence of alternative domestic animal models or that the domestic animal model has not been developed to the point where the key questions can be addressed. The study of the equine lentivirus EIAV as a model of HIV [114, 115], Anaplasma and Babesia in cattle as models of vector-borne pathogens [116-118], and scrapie in sheep as a model of spongioform encephalopathy [119] are only a few examples of projects that have provided novel insight into human diseases. In addition, the cow is an underutilized model for uterine infection and immunity [120]. Enhanced research into domestic animal models of infectious disease is necessary to expand our knowledge of fundamentals in transmission and pathogenesis of infectious agents.


Rotavirus causes severe gastroenteritis in infants and young children and animals worldwide. Gnotobiotic pigs are excellent models because their gastrointestinal physiology and development of mucosal immunity and susceptibility to infection with human rotavirus strains resembles humans. In addition, the pigs are born virus-free and without maternal antibodies, which eliminates confounding variables and enables assessment of true primary immune responses [121].

Vaccine development: Development and testing of novel vaccines in domestic animals is both informative as models and as a means to directly control spread of zoonotic diseases. Domestic animals respond to vaccination with marked variation in responses among individuals, similar to the responses of humans but unlike inbred mouse models [122, 123]. This variation in response allows testing of novel vaccine constructs to enhance responses of individual “poor responders” as well as to examine the effects of this variation in immunity among individuals at the population level. Both issues are of fundamental importance in improving vaccination as a means to improve human health. In addition, the development and deployment of animal vaccines have been used to directly control disease spread to humans, the most striking example perhaps being rabies virus, in which human health is protected essentially entirely through vaccination of animals, rather than humans themselves [124]. This concept is applicable to a variety of food and water-borne pathogens, vector-borne diseases, and may extend to prevention of transmissible spongioform encephalopathies (prion diseases) [125, 126].


Table 1. Research areas (not prioritized) that potentially could be advanced by use of agricultural animals as biomedical models. ______________________________________________________________________

Epigenetics and environment: effect of photoperiod, global warming, seasonality, and elevation on modification of gene function

Reproduction: gametogenesis, gonadal function, infertility

Aging: skeletal diseases, especially chicken and pig models; bone metabolism and osteoarthritis, especially the horse model; reproduction, especially beef cattle, mares

Obesity: genetic, dietary, hormonal influences on pre- and post-natal adipose tissue development using pig model [86, 89]

Pregnancy: placental growth, angiogenesis, congenital and birth defects, developmental biology especially chickens, fetal programming especially sheep to study stress, malnutrition, effects of exposure of fetuses to androgens and environmental toxins on adults, molecular/cellular basis of parturition and premature birth

Diabetes Types I and II [70].

Therapeutics: xenotransplantation [11, 127], gene therapy, pharming [91, 127], stem cells, “Farmaceuticals” [3]

Toxicology [128], environmental endocrine disrupters

Neurobiology: behavior, stress, learning, pheromonal communication, neuroendocrinology

Immunology: autoimmune disease, inflammation, innate and mucosal

Cardiovascular disorders such as diet-induced artherosclerosis [86], and lethal cardia tachyarrhythmias (ventricular fibrillation) using miniature or normal pigs

Nutrition: energetic balance including homeostatic mechanism, regulation of metabolism, use of neonatal piglet as pediatric model for studies of nutrition,metabolism and gastroenterology (see references in:[89])

Ophthalmology: retinal degeneration, retinitis pigmentosa [129], macular degeneration

Comparative physiology (e.g., Understanding of what makes cattle breeds different with respect to reproduction, lactation, growth, bone structure, fat deposition, altitude and heat tolerance, and resistance to specific pathogens will be invaluable in elucidating related physiological processes important to human health)

Interventional Radiology [130]

Biomechanics

Renal biology

Diseases: Transmissible Spongiform Encephalopathies (TSE); Respiratory Syncytial Virus (RSV); Crohn’s Disease; sexually transmitted diseases (STD); enteric including Transmissible

Gastroenteritis (TGE); viral, E. coli 01578; cancer including prostate, breast, ovary (chicken), hematopoiesis, leukemia; cattle as a model for salmonellosis, tuberculosis and cryptosporidiosis; pathogen transmission of emerging diseases that infect animals and humans such as use of cattle to study tick-borne infections

Disorders: liver, epilepsy, and sleep such as narcolepsy

Microbial ecology

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