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6 Technologies for Improving Animal Health and Production Roles of Animals in Society The importance of livestock in the economies of developing countries can be measured in terms of human health, as a contribution to gross domestic product, as a pathway out of poverty, and as a buffer against unforeseen disasters often faced by small-holder farmers. In addition to the direct financial benefits they provide, animals play many roles for small- holder farmers: they provide food, manure (as a soil amendment or fuel), traction, a savings mechanism, and social status (Randolph et al., 2007). When household members on crop-livestock farms in western Kenya were asked why their incomes had risen above the rural Kenyan poverty line (US$0.53/day), the top three reasons given were off-farm employment of a family member, production of cash crops, and livestock acquisition (Krist- janson et al., 2004). More than half the farmers who remained poor cited funeral expenses that typically involved the slaughter of livestock. Animals are perceived by farmers to be a useful hedge against drought, pests, and health problems. However, many farmers appear unwilling to sell animals even in stressful times, so economists debate whether they truly serve as a financial buffer (Dercon, 1998; Fafchamps et al., 1998). Animal protein plays an important role in human nutrition in sub- Saharan Africa (SSA) and South Asia (SA). Small amounts of meat or milk added to typical diets of Kenyan primary-school children increased their performance in school, improved results on cognitive-ability tests and ac- tivity levels, and reduced the incidence of stunted growth (Neumann et al., 2003). 177
178 Emerging Technologies to Benefit Farmers This chapter discusses the farming systems in which animals are pro- duced in SSA and SA and describes technologies for improving the health nutrition of food animals, the genetic foundation of food animal herds, and the protection of animals against disease. Animal Production Systems Animals are raised in several production systems in SSA and SA, each of which is constrained in different ways. Interventions to improve livestock (and the welfare of farmers) in each system must take into account the na- ture of its relationship to sources of animal feed or forage, the availability of food-processing facilities, and access to the intermediary or direct con- sumer markets for meat and dairy products. System modeling may be one way to envision the effects of interventions, not only on improving animal productivity but also on increasing income, reducing poverty, and prevent- ing environmental damage associated with livestock production (Box 6-1) (Charles Nicholson, Cornell University, presentation to committee, October 15, 2007). Of the 687 million poor who own livestock, 20 percent produce them in extensive systems (see Box 6-2), 57 percent live on mixed crop-livestock farms, and 23 percent are landless, peri-urban producers (Devendra et al., 2005). In Asia, more than 95 percent of the ruminants and many swine and poultry are raised on small crop-livestock farms. Those operations typi- cally are land-constrainedâoften less than a hectareâso feed availability is likely to pose a problem. Well-managed crop-livestock farms can take advantage of the value added by livestock by using manure to prevent soil- nutrient depletion. As the demand for meat increases, mixed crop-livestock farms are intensifying and increasing the risks of environmental problems associated with agriculture. The landless livestock systems, mostly in peri-urban areas, typically include swine, poultry, and ruminants. Because very little land is involved, less than 10 percent of the feed resources are produced where the animals are housed (SerÃ© and Steinfeld, 1996). The peri-urban farmers, especially those raising poultry and swine, rely more on concentrates for feeds and are likely to be adversely affected when grain prices increase, for example, in response to international demand for biofuels. The use of byproducts from processing human food could potentially provide valuable feed resources for these farmers. There is a need for improved food processing capabilities (see Box 6-3) that would help small farmers to have better access to markets (Delgado, 2005). The accumulation of nutrients (animal waste) often leads to con- tamination of water supplies and health issues related to animal density and proximity to people; the use of animal waste for biogas production would
Technologies for Improving Animal Health and Production 179 BOX 6-1 Environmental Effects of Livestock Production A 400-page review of livestock production in the developing and developed worlds describes its associated environmental effects, including land degrada- tion, contributions to greenhouse gas production (carbon dioxide, CO2; methane, CH4; and nitrogen dioxide, NO2), depletion and contamination of water supplies, spread of zoonotic diseases through poor animal and manure management, and reduction in biodiversity (Steinfeld et al., 2006). The extent to which livestock contribute to environmental problems depends on the production system, but a comprehensive evaluation of livestock production systems is needed, including marketing, land tenure arrangements, and germane policies to minimize adverse effects. Scenario testing of alternative strategies will require robust models to evaluate the effects of changes in management, marketing, and policy. For example, it is estimated that livestock contribute about 9 percent to total anthropogenic CO2 production and 35 to 40 percent of CH4 emission, including those from enteric fermentation, manure management, and livestock-associated fertilizer application, tillage, feed processing, and transportation (Steinfeld et al., 2006). Effective manure management and use of biogas may reduce emissions by as much as 75 percent in warm climates. However, manure gas emissions are affected by temperature, moisture, animal diet, physiological status of the animal, and storage methods, so current predictions of emissions all have huge coeffi- cients of variation. Both data and appropriate models are needed to predict which parts of the system are amenable to useful manipulation to decrease greenhouse gas emissions. be an area worth further exploration. In many cases, the term peri-urban is essentially used to describe agriculture in the slums, where poor sanitation, disease, and non-potable water already pose serious problems. Peri-urban swine and poultry systems are already a major source of food for city dwell- ers, but because of the hefty initial capital requirements and environmental considerations they are unlikely to become pathways out of poverty. Improving Animal Nutrition Many of the animals raised by small farmers in SSA and SA suffer from poor nutrition. As a result, they grow slowly, produce small amounts of milk or meat, have low reproductive rates, and are vulnerable to disease, even from birth. This section discusses some current and emerging oppor- tunities to decrease mortality in young livestock and improve the nutrition of farm animals.
180 Emerging Technologies to Benefit Farmers BOX 6-2 Animal Production in Extensive Rangeland Systems In many semi-arid and arid areas in Africa where rainfall is insufficient for reliable cereal harvests, animals contribute more than 80 percent of the agricultural GDP (Winrock International, 1992). The extensive livestock systems in the dry lands pro- duce about 10 percent of the global meat supply, but the areas where these systems dominate make up about one-fourth of the worldâs land mass, and low rainfall makes crop production impossible or very risky. Livestock production is the primary food- and income-generating activity for the people in those areas. When market values were assigned to home-consumed goods in a study of the Gabra of northern Kenya, 76 percent of the total was from home-consumed milk and meat, and 21 percent was from goods purchased with revenue from livestock sales, so only 3 percent of household consumption was from non-livestock sources (McPeak, 2003). More people in the world depend on sheep and goats for their survival than any other species; these small ruminants therefore play an important role in the sustain- ability of humans. Many parts of SSA either do not have the grain-based diets for com- mercial production of monogastrics such as chickens and pigs or may have religious practices that are not conducive to the development of a swine industry. Also, the lack of disease surveillance, particularly in poor rural areas, makes it difficult to raise swine and poultry in the countries most affected by disease. Consequently, increasing the knowledge of the genetics, physiology, breeding, nutrition, and diseases of small ruminants and the social structures of pastoralism is of paramount importance. It can be argued that the populations of pastoralists and herders are small and that develop- ment resources would be better spent elsewhere. That choice would be devastating to the herding populations of Niger, Burkina Faso, Mali, Chad, and Ethiopia, the five Reducing Preweaning Mortality A nutritional intervention for decreasing mortality in young livestock would include the use of colostrum for neonates. Colostrum contains high levels of energy and nutritionally important proteins, and neonates depend on the early infusion of nutrients to maintain body temperature because their energy stores are low at birth. More information on neonatal passive immunity is provided later in this chapter. Improving Grass Forage Many forage-fed animals in the tropics grow slowly and produce small amounts of milk because their diets are inadequate in protein, energy, and micronutrients. The types of forage available to the animals are mainly the
Technologies for Improving Animal Health and Production 181 countries ranked at the bottom of the UN human development report in 2005. Likewise, drier areas of central and southern Asia, where livestock dominate, suffer from extreme poverty. Dryland livestock production is heavily affected by low and variable rainfall and is thus vulnerable to the effects of climate change. In harsh environments, survival of animals and their owners depends on access to somewhat less marginal areas for part of the year. Changes in land tenure and increases in protected areas have decreased the mobility needed to avert overgrazing and to obtain access to feed reserves during dry seasons. Suboptimal distribution of animals results in localized degradation sur- rounded by range that remains productive. Post-drought restocking programs often provide families that have lost all their animals with one or two replacements despite data from northern Kenya and southern Ethiopia that show that at least five cattle are needed for self-sufficiency (McPeak, 2003). Nutritional inadequacy is a severe seasonal constraint in dry areas, but pasture improvement of extensive land systems is extremely difficult. Farmers lack the equip- ment and improved forage varieties needed for pasture establishment, and free-ranging animals often consume newly planted pastures. Although there is potential to improve livestock productivity in dry areas, many of the most feasible solutions involve inte- grated applications of current knowledge rather than new technologies. Biophysical and socio-economic models that include policy considerations that influence rangeland productivity could be used to predict effects of fluctuations in herd sizes, rainfall, and land tenure. Early warning systems and drought predictions could benefit herders in extensive systems provided they were mobile enough to access reserve pastures or were willing to sell stock. Efforts of that sort are under way, but more comprehensive data and better modeling techniques are both needed. C4 grasses (so named for the metabolic pathway used to fix carbon diox- ide). The C4 grasses that predominate in the tropics are less digestible than temperate C3 grasses and have low energy and protein content. As the data in Figure 6-1 show, only 45 percent of the 80 tropical feed samples tested met the maintenance requirements for both protein and energy and 30 per- cent of the samples were inadequate in both (Van Soest, 1994). Until recently, there was little work on characterization of forage traits that need improvement, and temperate forage still receives far more atten- tion than that grown in the tropics (Spangenberg, 2005; Smith et al., 2007). In general, tropical forage plants have received little attention from plant breeders with a few notable exceptions: alfalfa, which is grown in some tropical highlands; Brachiaria spp.; Pennisetum purpureum (elephant or Napier grass); and Panicum maximum (Guinea, colonial, or Tanganyika grass (Jank et al., 2005).
182 Emerging Technologies to Benefit Farmers BOX 6-3 Food Processing and Production Post-harvest mishandling of animal products results in substantial product losses and health hazards due to foodborne disease, but lack of refrigeration, inadequacy of fly control, and contaminated water supplies make preservation of highly perishable products difficult. Because risk of contracting foodborne disease is higher by as much as 3,000 percent in malnourished populations (Morris and Potter, 1997), appropriate control is particularly important in food-insecure areas. There are opportunities for local improvements in preserving meat, milk, and fish. In developing countries, people rely on drying, salting, and fermentation for food preservation because of capital constraints and lack of electricity, whereas people in developed countries depend more on refrigeration, canning, freezing, dehydra- tion, and fermentation. Traditional fermentation is used widely around the world (Steinkraus, 2002), but there are promising improvements in the use of bacterial inocula that have nutritional benefits or that stimulate production of bacteriocins to reduce microbial contamination. Amino acid profiles and contents of vitamins and protein may be improved through fermentation. Much is known about these technologies, but adaptive research is needed to ensure their effectiveness in low-resource environments. Investment in interdisciplinary adaptive research in African or Asian universities would both provide better methods to preserve animal-source foods and address the pressing need for more people with exper- tise in food safety. Such research should include the Hazard Analysis and Critical Control Point approach, which focuses on areas where significant improvements can be made. The collections of germplasm of tropical forage are poorly funded, and loss of current accessions is threatened. The collections include diverse ac- cessions that may be important sources of disease resistance, increase in di- gestibility, or increase in biomass production. For example, the most recent outbreak of a smut (Ustilago kamerunensis) is affecting Napier grass. In much of eastern and southern Africa, farmers rely heavily on Napier grass because it produces copious amounts of reasonably high-quality forage. The smut has the potential to affect the small-holder dairy industry seriously and has already reduced forage yields in much of the Kenyan highlands (Farrell et al., 2002; Mwendia et al., 2007). Research to understand smut biology and to develop resistant strains of Napier grass is important for the rapidly growing dairy industry in the Kenyan highlands. As noted in Chapter 3, a better understanding of plant chemistry and lignin synthesis could help plant breeding programs to improve the nu- tritional value of forage because the lignin cross-linkages affect whether
Technologies for Improving Animal Health and Production 183 FIGURE 6-1â Digestibility and crude protein content of tropical grasses (fertilized 6-1.eps and unfertilized) and legumes and their adequacy in meeting maintenance require- ments of ruminants. bitmap image NOTE: 52 percent dry matter digestibility and 8 percent crude protein. SOURCE: Reprinted from Peter J. Van Soest: Nutritional Ecology of the Ruminant, Second Edition. Copyright Â© 1982 by P. J. Van Soest. Copyright Â© 1994 by Cornell University. Used by permission of the publisher, Cornell University Press. plants are easily digested (Spangenberg, 2005). There may be advantages in attaching work on this problem to the burgeoning international interest in biofuels, such as switchgrass. There is a common interest in understanding how lignin cross-linkages can be broken down, whether in the context of biofuels or with respect to the processes occurring in forage digestion by ruminants (Box 6-4). Improving Legume Forage In temperate areas, legumes, especially alfalfa and clover, are high- protein, highly digestible forage that permit cows to sustain milk produc- tion as high as 20 kg/day on forage alone. In the tropics, however, many
184 Emerging Technologies to Benefit Farmers BOX 6-4 Rumen Function, Fiber Digestion, and Metagenomics Ruminants will probably be important in livestock strategies to assist the poor (Delgado, 2005), therefore their ability to convert locally available feedstuffs to animal products should be improved. Increasing the efficiency of the ruminal mi- croorganisms that play important roles in fiber digestion and nitrogen metabolism will improve animal productivity. When Hungate (1966) published The Rumen and Its Microbes, about 23 bacterial species were thought to play prominent roles in ruminal metabolism; by 1996, the number exceeded 200 (Krause and Russell, 1996). When several discrete ribosomal DNA libraries were analyzed, 341 operational taxonomic units of organisms were identified (Edwards et al., 2004); this indicated that culture-based estimates of ruminal organisms greatly under- estimated ruminal diversity. Simple identification of individual species is far less important than understanding the functions of microbial populations and relating them to sequence-based information to draw ecological inferences (Handelsman, 2004). The recent study of gypsy moth gut microflora that included quorum sensing, the coordination of biological functions among bacteria, and identification of cell signaling mechanisms (Guan et al., 2007) is an example of the type of research needed to improve ruminal fiber digestion and nitrogen metabolism and to re- duce methane production. Because the techniques needed for the study of the rumen are similar to those required for the study of other microbial systemsâ including soils, food fermentation (such as that of yogurt and cheese), and biofuel productionâemphasis on various methods for studying microbial ecology would have broad benefits. Not only is ruminal microbial diversity much greater than early estimates suggested, but the enzyme systems involved in lignocellulosic degradation are much more complex (Huang and Forsberg, 1990; White et al., 1990; Bayer et al., 1998). Each bacterium has many types of enzymes (for example, endogluca- nases, exoglucanases, cellobiohydrolases, and xylanases) and many enzymes with overlapping activities. Our knowledge of the enzymes remains incomplete, and novel hydrolases are being discovered (Ferrer et al., 2005). In some cellulo- lytic anaerobic bacteria (such as Clostridium thermocellum, Ruminococcus albus, and Ruminococcus flavefaciens), the diverse enzymes are organized by scaffold- ing proteins into cellulosomes in which dockerins permit substrate binding and efficient cellulose degradation (Bayer et al., 2004). Much has been learned in the last 20 years about the functions and organization of cellulosomes and their many enzymes, but this remains a fertile field of inquiry. Recent advances in genomics and proteomics should assist in the research, but the inability to transform and genetically manipulate ruminal microorganisms constrains progress.
Technologies for Improving Animal Health and Production 185 promising legume species contain high concentrations of anti-nutritional factors (such as proanthocyanidins, hydrolyzable tannins, alkaloids, and terpenoids) that confer disease resistance on the plants and deter herbivory. Condensed tannins have both beneficial and deleterious effects on domestic animals (Mueller-Harvey, 2006). The adverse effects of consum- ing high-tannin forage include lower feed intake, lower protein and dry matter digestibility, inhibition of microbial and mammalian enzymes, re- duced live weight gain and milk yield, and systemic effects that are due to absorption of phenolics and are sometimes offset by lower urinary nitrogen loss, greater parasite resistance, and improved efficiency of nutrient use (Mueller-Harvey, 2006). The apparently contradictory research results are due largely to the heterogeneity of tannin structures and to variation in the quantities ingested. Achieving the goal of developing disease-resistant legumes that provide animals with needed nutrients requires research on tannin chemistry linked to legume breeding programs. Progress has been made in understanding some aspects of tannin synthesis, but the polymerization process that affects tannin chemistry and anti-nutritive effects remains poorly understood (Xie and Dixon, 2005). Existing and evolving Technologies For Improving Animal Germplasm Since the beginning of domestication of animals, substantial progress has been made in improving their characteristics as food and fiber produc- ers by selectively mating individual animals that had advantageous traits (phenotypes). The importance of phenotypic information is sometimes lost in this age of genomics, and it is astonishing to recognize that animal breeders could triple average milk yield of dairy cattle in 50 years without knowing a single gene involved or having any genome sequence information to guide them. They simply needed to know the milk production traits of members of the dairy cattle family and select the right mates to breed. Although it is possible to practice breeding of that type on a farm or village scale, small-herd owners in SSA and SA are likely to have difficulty in systematically improving the genetic potential of their livestock by us- ing only locally available germplasm. Nor can small-holders apply modern quantitative breeding practices on the basis of the knowledge of genotypic associations with specific traits; information systems to collect phenotypic and genotypic data from populations of the desired species or breeds sys- tematically have not been put into place. The use of quantitative phenotypic methods to improve breeding re- quires collecting data on a large number of animals in a family that exhibit
186 Emerging Technologies to Benefit Farmers BOX 6-5 Genetic Improvement of Fish for Aquaculture Fish provide substantial amounts of protein and other frequently deficient nutrients in Asia and parts of Africa. In SSA and SA and globally, aquaculture is growing in economic and nutritional importance. The World Fish Center (2005) recently identified strategies for aquaculture development with representatives of Bangladesh, China, India, Indonesia, Malaysia, Philippines, Sri Lanka, Thailand, and Viet Nam. The use of molecular markers associated with genetic improve- ment of fish, including disease resistance, has long been recognized as feasible (Austin, 1998), and one can identify a wide array of disease susceptibility in fish populations (Kettunan et al., 2007; Quillet et al., 2007). The development of inbred lines and the use of markers for selection are meeting with success (Gilbey et al., 2006; Zhang et al., 2006). wide variation in the traits of interest. That kind of effort typically takes place in breeding centers, where resource populations of animals can be developed over a decade or two and individual phenotypes can be collected and recorded to make it possible to identify genetically superior animals (Meuwissen and Goddard, 2000, 2001). Infrastructure is needed to distrib- ute the germplasm to farmers through artificial insemination and embryo transfer techniques. In industrialized countries, that approach has been used over the last 50 years to develop animals with superior genetics, and it is the model used in developing countries, often successfully (Box 6-5). However, the scientific community is now in a position to bypass many of the heavily resource-dependent approaches used in the industrialized world. Emerg- ing technologies offer potentially practical approaches for more rapidly discovering superior livestock genetics and delivering them to subsistence farmers in SSA and SA. Leapfrogging Selective Breeding with Molecular Sampling: DNA-Derived Pedigrees There are about 170 million buffalo (Bovidae) in the world, 96 percent of which are in Asia, including 95 million in India alone (Borghese, 2005). The American bison is also included in that estimate. The current status of buffalo production and research has recently been described in detail (Borghese, 2005). The buffalo is a primary source of milk protein and is
Technologies for Improving Animal Health and Production 187 used for draught and as a supplementary source of meat in parts of Asia. Ten major breeds exist in India, some having been selected and maintained for each of the three functions, which are essential to the farm economy. National programs to improve milk and meat production have been initi- ated and are being termed the âwhiteâ and âredâ revolutions, respectively, in keeping with the name of the Green Revolution. Genetic improvement for production traits and disease resistance in buffalo does not benefit from the availability of the powerful genomic tools recently generated for domestic cattle in the United States, for two main reasons: the areas of the world where buffalo are economically important lack the financial resources for genomic research, and the application of genomic research to identify genetically meritorious individual animals can be applied only within families of animals. There is no such information on Bubalus bubalis, the Asian water buffalo, or on any farm animals (such as goats and hair sheep) raised by subsistence farmers in SSA and SA, so the use of well-established quantitative genetic tools is precluded. However, it may be possible to construct an equivalent dataset from the bottom up with the aid of molecular genetic tools. To implement that ap- proach, a reference genome of the breed of interest would need to be gener- ated with DNA sequencing, and DNA samples and phenotypic data would have to be collected from several thousand animals in geographic regions that have common environmental stresses. Single nucleotide polymorphisms (SNPs) would be generated from the DNA samples by sequencing regions of the genome that have proved to be informative in related species. The database of tag SNPs generated from the sequencing data would be aligned with the reference sequence to build family pedigrees. With pedigrees in hand, traditional quantitative tools could be applied to identify animals of superior genetic merit. The approach requires several lines of research. An inexpensive field kit for preserving DNA in tissue samples (ear snips, buccal swabs, or the equivalent) that does not require refrigeration would have to be developed, as would an effective questionnaire for gathering trait phenotypes. Whole genome sequencing (6X coverage) of Bubalis Bubalis, the African buffalo (Syncerus caffer), Bos indicus cattle, sheep, and at least a few representative milk- and meat-producing breeds of goats and sheep should be included in the sequencing project. Finally, substantial invest- ment in developing informatics algorithms for what is essentially reverse engineering of pedigrees from SNP data would be needed. With todayâs sequencing capability, it might take 2 years to generate a reference genome sequence for a species. It might take a year each to de- velop a DNA tissue-sample preservation kit and a phenotype questionnaire, 3 to 5 years to collect DNA samples and phenotypic data, and a year to build pedigrees and test the hypothesis that animals of high genetic merit can be identified with this approach. All the steps except the last can be
188 Emerging Technologies to Benefit Farmers conducted in parallel, the overall timeframe of the project to reach proof of concept would be 6 to 10 years. If the project were successful, its impact would be large. DNA-enabled approaches to building a pedigree would leapfrog existing approaches by eliminating the decades of breeding needed to create resource popula- tions, the usual starting point of contemporary genetic-genomic analyses. The substantial costs of housing and feeding such a population would be eliminated. Most important, it would provide a tool to identify genetically superior animals without having to develop the enormous infrastructure currently used in the developed world. Genetic Engineering For the first 8 to 10 millennia since animal domestication began, se- lective breeding has been the method by which desirable phenotypes were enriched in a population. The dramatic diversity generated in dog breeds and improvement in the efficiency of producing dairy cattle are just two examples of the power of selective breeding (Weller, 1994; Pennisi, 2007). However, the approach has limitations, of which the largest is the inability to introduce a trait if genetic information on the trait does not exist in the species of interest. For example, endowing swine with the ability to synthesize lysine de novo, which would eliminate the need to supplement feed with what is now an essential amino acid, is impossible because the biochemical pathway does not exist in any breed of swine. The pathway does exist in bacteria and yeast, but that is of no use to the animal breeder. Furthermore, selective breeding lacks precision. There are many examples of selecting for one important economic trait at the expense of another, such as sacrificing the reproductive performance of dairy cattle for increased milk production (Wicks and Leaver, 2004). But, as previously described, the most serious constraint in applying modern tools of genetic selection is the time needed to build resource populations and harvest the phenotypic information needed to populate the genetic algorithms for each breed of interest. In 1981, a new method for altering the genetic makeup of mammalian offspring led to the ability to place genes from one species (transgenes) into the genome of another (Gordon and Ruddle, 1981). Transgenes can encode completely novel natural or synthetic information, modulate the level of gene expression, or switch transcription on or off conditionally or permanently (Niemann and Kues, 2007). In the last 2 decades, geneti- cally engineered cattle, chickens, goats, pigs, rabbits, and sheep have been produced (Hammer et al., 1985; Salter et al., 1987; Bondioli et al., 1991; Ebert et al., 1991; Krimpenfort et al., 1991). Transgenic livestock applica- tions have been diverse and range from projects focused on animal well-
Technologies for Improving Animal Health and Production 189 being (Wall et al., 2005) to drug manufacturing (Edmunds et al., 1998). Transgenic animal technology has now advanced to the point where specific genetic information can be introduced precisely into any desired location of the genome (Richt et al., 2007). In theory, the technology provides a vast array of new ways to address challenges in animal agriculture. Engineering Animals for Disease Resistance Transgenic technology can be used in many ways to reduce susceptibil- ity to disease in animals. It can be directed at a specific pathogen or a wide variety of pathogens, depending on the protein encoded by the transgene. There are dozens of examples of successful application of genetic engineer- ing to protect mice, and many are expected to be predictive of outcomes of transgenic livestock experiments. However, to date there is only one example of genetic engineering that has protected a livestock species from disease (Wall et al., 2005). In general, a transgenic strategy for disease resistance involves identify- ing an anti-pathogenic protein to be expressed in the animal and determin- ing in which tissue and at what developmental stage expression should occur. The transgenic protein should cause no harm to the animal itself or to the consumer that eats it. If possible, the transgene product should avoid interfering with endogenous homeostatic feedback loops. The recombinant protein produced should be benign to the environment. Finally, a plan should be devised to prevent the target pathogen from developing resistance to the transgene product. Box 6-6 describes a project of this nature that would be considered promising but unprecedented and is of moderately high risk. RNA Interference RNA interference (RNAi), which is also discussed in Chapter 3, is an evolutionarily conserved mechanism of plants and animals that processes microRNA (miRNA) and destroys double-stranded RNA, targeting, in a sequence-specific manner, both messenger RNA and retroviral genomes (Hannon, 2002; McCaffrey et al., 2002). Theoretically, it should be pos- sible to target any virus with this mechanism. Retroviruses, with their RNA genomes, are an obvious potential target (see Box 6-7), but DNA viruses could be targeted if their provirus encodes a unique mRNA that could serve as a target. The RNAi approach to targeting HIV-1 infection has been demonstrated in cell-culture studies (Anderson and Akkina, 2005). And RNAi approaches have been devised to inhibit viral evolution of resistance (Anderson et al., 2007). The idea of using RNAi as a viral therapeutic is not new but has
190 Emerging Technologies to Benefit Farmers BOX 6-6 Engineering Chitinase as an Insecticide It may be possible to use transgenic technology to protect animals simulta- neously against a variety of vector-borne parasitic diseases, such as trypanoso- miasis (carried by tsetse flies), tick-borne East Coast fever, and Rift Valley fever (carried by mosquitoes). The strategy is based on attacking the carrier insect by disrupting chitin, an abundant N-acetyl-Î²-D-glucosamine polysaccharide that serves as a protective structural component of its exoskeleton. The transgene product, chitinase, which hydrolyzes the Î²-1,4 linkages in chitin, would be ex- pressed from the transgenic animalâs hair follicles. This approach, which uses chi- tinase as an insecticide, has been demonstrated in transgenic plants (Ding et al., 1998; McCafferty et al., 2006; Vellicce et al., 2006; Funkhouser and Aronson, 2007). Furthermore, hair follicle-specific promoters, such as keratin, have been shown to express transgene products in a tissue-specific manner in genetically engineered animals and in a gene therapy context (Powell et al., 1983; Ward and Brown, 1998; Paus and Cotsarelis, 1999). A proof-of-concept transgenic mouse experiment would need to be conducted to determine whether the strategy is safe and effective. Transgene design opti- mization to improve efficacy might be required. To create a herd of transgenic animals, knowledge of the reproductive physiology of the breed of interest must be known. When a herd is produced, an experiment should be conducted to test the hypothesis that expression of the transgene is protective; if it works, it will be necessary to begin a second phase of the project designed to discover at least two more anti-insect vector peptides (perhaps antibodies to insect gut organisms or proteins) that can be used in conjunction with chitinase to reduce the likelihood that insects would become resistant to the strategy. Overall, proof of concept in a livestock species would take at least 15 years for water buffalo and somewhat less for small ruminants because of the difference in generation intervals. Discovery and testing of second-phase anti-insect vector peptides may require another 10 to 15 years. Insect vector-borne diseases have been the focus of research efforts for nearly 100 years. Classically, each disease is studied in isolation primarily because each disease has a fairly unusual set of circumstances. If livestock could be protected against these insect vector-borne diseases, animal lives would be saved, and vast regions of western Africa that are now restricted by such diseases could be opened to livestock production. not been fully explored, possibly because of a number of potential hurdles (Silva et al., 2002) and because the focus has been on using the technology in basic research (Hannon and Rossi, 2004; Silva et al., 2005). But it is clear that targeting gene expression can be achieved with this approach in mammals (Kunath et al., 2003; Dann et al., 2006), and RNAi has recently been shown to work against influenza virus in mice (Zhou et al., 2007). As
Technologies for Improving Animal Health and Production 191 BOX 6-7 RNAi Technology to Resist Bluetongue Virus Bluetongue is a non-contagious, insect vector-borne viral disease of domes- tic and wild ruminants. In India and parts of Africa, bluetongue virus (an RNA orbivirus) is endemic; cattle and wild ruminants serve as reservoirs for the virus. Sheep are the ruminants most susceptible to the virus, which attacks the animalâs vascular system, sometimes causing the tongue to appear blue, and results in death within 2 to 5 weeks of infection (OIE, 1998). Transgenic animals resistant to the bluetongue virus would be produced by introducing a transgene that encodes several 19- to 25-base-pair inverted nucleo- tide repeats and creates short double-stranded RNA hairpins (shRNA) under the control of constitutive promotersâsuch as cytomegalovirus, ubiquitin C, or U6 promotersâin a lentivirus vector. Cell-culture studies followed by transgenic mice studies would be used to assess and optimize construct design before mov- ing into a livestock species. All the components needed to conduct such a study seem to be available. The therapeutic use of RNAi in livestock would be novel. Bluetongue is sug- gested as a target because it is not a communicable disease, so challenge studies are easier to undertake. In addition, the complete sequence of the bluetongue virus is known (NCBI, 2008). The overall proof of concept for using RNAi in a small ruminant would take about 10 years, including the design of an optimized transgene construct, cell- culture and mouse experiments, gamete harvesting, and production of transgenic livestock. Once an adequate number of transgenic and control animals are avail- able, infection studies to test the hypothesis will be needed, and additional time will be necessary to demonstrate that resistance does not develop. Introgressing the transgenes into a wider population through conventional breeding will take additional time. If successful, this project would serve as a new paradigm for prophylactic treatment of viral disease for which vaccines or other approaches are ineffectual or too expensive. Furthermore, the understanding of the reproductive physiology of the indigenous breed tested and knowledge about the shRNA transgene design will make future targeting projects less expensive. with any small interfering RNA project, it would be necessary to monitor potential off-target effects (Schramke et al., 2005). Fundamental Research Needed for Genetic Engineering Before genetically engineered animals can be produced, it is neces- sary to have a precise understanding of the reproductive physiology of the breeds of interest and protocols for processing their gametes and embryos.
192 Emerging Technologies to Benefit Farmers Optimized procedures will have to be developed for estrus synchronization, superovulation, ovum pickup, gamete preservation, in vitro fertilization, embryo culture, embryo manipulation, and somatic cell nuclear transfer for each of the local species of interest. It is not enough to have a general understanding of the species of interest, because subtle breed differences can often result in suboptimal yields. Most of the economically important animals of SSA and SA are related to species that have been the subjects of intensive scientific investigations in the agricultural setting of Europe and North America. Furthermore, initial studies have been conducted on indigenous species. Therefore, to develop the baseline technology needed to introduce new genetic informa- tion directly into the animals of interest, researchers can take advantage of experimental designs and nominal parameters established in European breeds of dairy and beef cattle, dairy and meat goats, and sheep. Advancing the technologies to the level of proficiency necessary to con- duct genetic-engineering experiments has other direct and immediate ad- vantages. Efficient procedures for collecting, manipulating, and preserving gametes and embryos can serve as the basis for distributing the best genetics of the day when the physical infrastructure and preservation methods are in place and genetically superior germplasm has been identified. A significant effort has already been made to apply strategies developed for Bos taurus (common domestic cattle of Europe) to the water buffalo. It is clear from the current scientific literature that the general characteris- tics of domestic cattleâs reproductive physiology are similar to those of the water buffalo, but specific differences require additional breed-focused re- search to optimize control of reproduction and in vitro viability of gametes and embryos (Saikhun et al., 2004; Boonkusol et al., 2007; Drost, 2007). Similarly, the procedures developed for European and Egyptian breeds of goats should serve as good starting points for the various commercially use- ful breeds of goats in SSA and SA (Armstrong and Evans, 1983; Cameron et al., 1988; Baril et al., 1989; Nowshari et al., 1995). Scientists in SSA, SA, and China are contributing to the scientific litera- ture that defines protocols for assisted reproduction technologies in goats (Espinosa-MÃ¡rquez et al., 2004; Hasin et al., 2004; lez-Bulnes et al., 2004; Goel and Agrawal, 2005). Germ Cell DISTRIBUTION No matter how the genetics of indigenous farm animals are improved, there needs to be a means of distributing the improved genetics to farmers. Whether by quantitative genetics (selective breeding) or transgenic technol- ogy, the germplasm of the lineage progenitors will be produced at a high- technology center that resembles a modern-day artificial insemination (AI) stud farm. A distribution system must be in place to allow farmers access
Technologies for Improving Animal Health and Production 193 to the superior genetic material; the lack of a distribution system seriously constrains the improvement of the genetic potential of subsistence farmersâ livestock. Since the 1950s, genetic improvement of the livestock herds in indus- trialized nations has been achieved primarily by distributing gametes (sper- matozoa) from outstanding sires and more recently by distributing embryos from meritorious females (Hasler, 1992; Foote, 1998; Thibier, 2005). As currently practiced, AI and embryo transfer (ET) require a ready supply of inexpensive liquid nitrogen. Liquid nitrogen must be available during the initial gamete- or embryo-freezing process and thereafter as a storage me- dium. On-farm storage dewars require replenishment (commonly every 4 to 6 weeks) that depends on use and environmental temperature. Furthermore, the use of preserved spermatozoa or embryos requires a specific knowledge of female reproductive physiology and highly skilled and practiced people to transfer the gametes or embryos into the recipient females. Even in de- veloped countries, AI and ET are practiced mainly in intensively managed livestock operations, such as medium to large dairy, swine, and poultry farms. Less intensively managed enterprises, such as beef-cow and -calf op- erations or small farms, do not use these reproductive strategies, primarily because of the cost of estrous cycle management, the lack of availability of skilled AI technicians, and the low return on investment. Attempts have been made to eliminate the dependence on liquid nitro- gen for preservation of gametes. At first, freeze-dried spermatozoa were not very successful (Norman et al., 1958; Foote et al., 1962; Wakayama and Yanagimachi, 1998). Recent refinements in freeze-drying protocols have resulted in live-born mice from spermatozoa stored for over a year at 4Â°C (Bhowmick et al., 2002; Ward et al., 2003; McGinnis et al., 2005). Such freeze-dried spermatozoa are not viable in the traditional sense, but viable offspring can be produced by injection of their nuclei into an oocyte (intracytoplasmic sperm injection, ICSI). Storage at â20Â°C or â80Â°C im- proves the success rate of ICSI over storage at 4Â°C (Li et al., 2007). Most recent attempts to adapt this technology to a non-rodent species have been unsuccessful (Meyers, 2006; Nakai et al., 2007). Modern cryobiology may eventually develop room-temperature storage methods that will preserve the genome of gametes. But it is also likely that any such technique will require some highly sophisticated method, such as ICSI, to introduce the stored genetics into a living organism. Such an approach would be impracti- cal for production of breeding stock. Spermatogonial Stem Cell Transplantation The use of an emerging technology called spermatogonial stem cell (SSC) transplantation may be able to overcome the infrastructure and technical skill deficiencies that will inhibit subsistence farmers from taking
194 Emerging Technologies to Benefit Farmers advantage of meritorious germplasm when it is available. SSC transplanta- tion (also known as male germ-cell transplantation or germline stem cell transplantation) involves transplanting self-renewing male germ-cell stem cells from one male to another. The recipient male becomes the mechanism for spreading the genetics through a herd. In the early 1990s, it was demonstrated that the stem cells that give rise to spermatozoa in a male mouse could colonize the testes of another mouse, and the recipient mouse could sire offspring with the donor-derived spermatozoa (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). The SSCs can also be frozen and stored at â196Â°C and, on thaw- ing, be used to colonize a recipientâs testes after transplantation (Avarbock et al., 1996). The technique also works in rats (Ryu et al., 2007), and it has been shown that it can be used to restore fertility in two mouse models of infertility (Ogawa et al., 2000). Most relevant here are reports that demonstrate the potential of ap- plying the technology to livestock. SSCs isolated from immature pig testes have been transplanted (Honaramooz et al., 2002), and the transplanted spermatogonia remained in the seminiferous tubules of the recipients for at least a month; somewhat surprisingly, not only did SSCs colonize the seminiferous tubules and generate spermatozoa capable of siring offspring, but this was possible in recipients unrelated to the host. Several studies also demonstrated the feasibility of the approach in goats (Honaramooz et al., 2003; Honaramooz et al., 2007). One attempt has been made to demon- strate its efficacy in cattle: testicular cells were isolated from Bos taurus bull calves and transferred, after fluorescent staining, into Bos indicus prepuber- tal recipient calves (Herrid et al., 2006); Bos taurus fluorescently labeled cells were found in the testes of recipients up to 6 months after transfer. It is envisioned that once this technology has been refined and adapted to local breeds, SSCs harvested from males with superior genetic merit will be distributed to males of average genetic merit but good libido. The recipi- ent males, harboring the âgood genetics,â could then be distributed to farm- ers. Alternatively, because the transplantation procedure can be performed in the field, the SSCs from genetically superior males could be frozen, transported to farms (or villages), and transplanted into farmersâ (villagesâ) own breeding males by someone skilled in the surgical procedure. The main constraint limiting the technology is the acquisition of enough SSCs. It is possible on rare occasions to harvest enough SSCs from a donor to distribute to four recipients (Ina Dobrinski, University of Penn- sylvania, presentation to the committee, October 15, 2007). Protocols are needed for multiplying SSCs in culture so that dozens, if not hundreds, of males can be serviced by a single genetically superior donor. In addition to a propagation system, there is a need for more efficient SSC-enrichment methods. Finally, methods to improve the ability of the newly acquired
Technologies for Improving Animal Health and Production 195 SSCs to dominate the testes of the recipient will need to be devised. The current literature on this new procedure is not vast, but there are enough examples to suggest that SSC transplantation should be applicable in a wide array of species. It will be necessary to confirm that SSC transplanta- tion can be implemented in the breeds and species of interest in SSA and SA and, if so, that it can be optimized for each species. Improving Animal health Improving the health of animals can have a substantial impact on the livelihood of farmers, especially subsistence farmers that rely on animals for labor, food, and additional income. Some of the ways to improve animal health discussed below include fortification of neonatal passive immunity, development of animal vaccines for diseases affecting SSA and SA, and use of animal disease surveillance. Not explored by the committee are the devel- opment of novel drugs and drug delivery strategies for animal disease infec- tions in SSA and SA, two areas in which innovations have been lacking. Neonatal Passive Immunity In Kenya, calf mortality after weaning ranges from 6 to 70 percent, depending on health and nutritional management (Homewood et al., 2006; Lanyasunya et al., 2006). Almost all the primary causes of high prewean- ing mortalityâincluding failure to ensure that the young receive colostrum within 6 hours of birth, respiratory diseases, diarrhea, and inadequacy of maternal milk productionâcan be substantially reduced with currently available, low-cost management interventions. These include giving young animals the same salt- and sugar-based rehydration solutions made with clean water as are given to children who have diarrhea. The application of existing knowledge to raising calves, lambs, and kids could reduce mortal- ity to below 9 percent, a commonly accepted target, and improve animal productivity and profitability. Technologies are used to enhance colostrum quality by vaccinating pregnant dams. Also, the preparation and preservation (freeze-drying) of serum antibody extracts are used as artificial colostrums substitutes. There are some truly nutritional interventions to prevent preweaning mortality, such as enhancing the nutrition of the lactating dam and providing nutri- tional supplements to the diets of lactating animals. The issue of access to veterinary services by the poor in SSA and SA is critical. The delivery of appropriate medicines and information on livestock health has been compromised by privatization of veterinary services in many countries. Now only those able to pay have access to veterinary ser- vices, so livestock productivity is low and the risk of outbreaks of zoonotic
196 Emerging Technologies to Benefit Farmers and other diseases is high. Very simple interventions in providing educa- tion, medicines, and vaccines could have a major impact in protecting the health and productivity of animal populations in SSA and SA. Reversing the situation would make meeting the projected increased demand for meat products more feasible without large increases in the number of breeding females. Animal Vaccine Development Disease is a major constraint on livestock productivity in developing countries. Chapter 2 describes the long list of disease problems in SSA and SA. Estimates of losses due to disease in the regions are not well quanti- fied, although one estimate of the annual economic loss due to animal diseases in SSA is around US$40 billion, or 25 percent of the total value of livestock production. There is a substantial worldwide effort to develop animal vaccines, and several major U.S., European, and Asian pharmaceu- tical companies are highly invested in food animal vaccine production. In fact, when the committee asked several experts what could have a major effect on improving the life of poor farmers, they noted that effective vac- cines already exist to prevent globally endemic disease, such as brucellosis, leptospirosis, and bovine virus diarrhea (Hans Draayer and Raja Krishnan, Pfizer Animal Health, presentation to committee, September 24, 2007). However, some factors that affect the use of current vaccines in SSA and SA include strain variations, costs of vaccines, and the need to have an effective cold-chain for transportation, marketing, distribution, and delivery to the animals in the field. Technologies to develop thermostable vaccines, such as the development of a thermostable attenuated vaccine for Newcastle disease in chickens in Australia and Malaysia, can compensate for the lack of a cold-chain. A focus on infections for which vaccines exist and on others that cause respiratory and intestinal diseases in young, preweaned animals could reduce mortality and improve productivity. The other two categories of opportunity identified by experts the committee consulted were zoonotic diseases, particularly those associated with foodborne illness, and endemic infections peculiar to SSA and SA (Guy Palmer, Washington State Univer- sity, and Roy Curtiss, Arizona State University, presentation to committee, September 24, 2007). SSA and SA are home to the most severe vector-borne diseases, includ- ing trypanosomiasis, babesiosis, and theileriosis (the last two of which are parasitic diseases spread by ticks). Diseases, such as trypanosomiasis, have been the subject of vaccine investigations for many years and have thwarted vaccine effectiveness because of the great variability of surface proteins of the parasite, which the organism is able to âswitchâ under the pressure of
Technologies for Improving Animal Health and Production 197 the host immune system. The greatest challenge for these vaccines is the discovery of antigens that will result in a protective immune response in the host. Such a discovery will be assisted by the complete genome sequences that have been completed for all six major vector-borne pathogens in the last 2 years, including Anaplasma marginale, Babesia bovis, Ehrlichia rumi- nantium, Theileria parva, T. annulata, and Trypanosoma bruci. For exam- ple, it is known that immunity to East Coast fever, caused by the tick-borne parasite T. parva, can be created by inoculating a host with sporozoites of the parasite in conjunction with long-acting oxytetracycline. However, until the T. parva genome was available, the antigens involved in the immune response were unknown. Using gene prediction methods, investigators were able to identify candidate genes in the genome that were associated with a secretion signal on the basis of the idea that secreted proteins would be the first to become associated with the host major histocompatibility complex apparatus. Screening of the protein products of those genes narrowed the search to the ones involved in establishing immunity and paved the way to future vaccine development (Graham et al., 2006). Zoonotic diseases were suggested as targets for disease control because of their implications for limiting the spread of diseases to humans and back to animals. Effective vaccines exist for some of the diseases, including bru- cellosis, salmonellosis, and listeriosis; but problems related to supply, cost, and delivery mechanisms slow their widespread use. Bacteria as Antigen Vectors Because they are so infectious, serotypes of some of the disease-causing organisms have been used, in attenuated forms, as packages to deliver anti- gens for several pathogens in âattenuated recombinant bacterial host-vector vaccine systems.â Experimental work with Salmonella typhi suggests that it could be developed into a vaccine to protect against hepatitis B virus, human enterotoxigenic Escherichia coli, Mycobacterium tuberculosis, Clos- tridrium, Yersinia pestis, and other pathogens (Curtiss, 2002). The use of genetic engineering methods has dramatically improved vac- cine production compared with conventional methods of developing live attenuated and inactivated pathogens. The basic strategy for developing bacteria-based vaccines is to transfect a bacterial vector, such as Salmonella or Shigella, with plasmids that express the antigen of interest and inject the transformed bacteria into the host. This system allows the delivery of mul- tiple antigens, and the resulting expressed antigens would elicit antibody production to protect against several diseases. Attenuated strains of Salmo- nella typhimurium have been used for delivery and expression of vaccine antigens in the mouse (Ashby et al., 2005). In all cases, specific antibody against the antigen is detected in host blood. Information on comparisons
198 Emerging Technologies to Benefit Farmers with conventional vaccines is not complete, but this approach offers po- tential help in preventing parasitic diseases for which it has been extremely challenging to develop effective vaccines. For example, attenuated Salmonella typhimurium transformed with the Plasmodium berghei CS (circumsporozoite protein) gene induced protec- tive cell-mediated immunity to sporozoites in the host. The transformants, used orally to immunize mice, colonize the liver, express CS proteins, and induce antigen-specific cell-mediated immunity, protecting mice against sporozoite challenge in the absence of antisporozoite antibodies. It has been established that immunization with CS proteins by injection does not offer protection against sporozoites. However, Salmonella as the carrier of the CS gene and later the expression of CS protein stimulated T-cell-mediated im- munity. It is worth investigating the possible use of a vaccine for controlling other parasitic diseases caused by trypanosomes and Leishmania. Another case study demonstrated that live attenuated Shigella flexneri strains act as vectors for the induction of local and systemic antibody responses against poliovirus epitopes (Levine, 2006). Poliovirus proteins (IpaC-C3 hybrid proteins) were expressed by recombinant plasmids in S. flexneri. Research in this definitive area will yield new vaccines against diseases previously deemed difficult. More development is needed before these vaccines are reliable in immune protection of the host. Plant-Based Expression System for Vaccine Development Genetic engineering also has made it possible to use plants as facto- ries for pharmaceutical protein production. Unlike bacterial cells, plants are capable of some post-translational modification and other assembly steps that are needed for biological activity in complex multi-component proteins, such as antibodies. The plants that have been successfully trans- formed include tobacco, potato, tomato, corn, soybean, alfalfa, rice, and wheat. The proteins made in plants have been used to produce antibodies, vaccines, hormones, enzymes, interleukins, interferons, and human serum albumins (Moschini, 2006). Plant-made biologicals are created by inserting into plant cells a seg- ment of DNA that encodes the protein of choice. The plants or plant cells are essentially molecular factories that can be used to produce the desired proteins and are grown only for pharmaceutical applications. In addition to vaccines meant for humans, plant-based vaccines are being developed for use in animal health. In fact, edible plant-based vaccines might be best suited for animal applications: an edible product can be conveniently added to animal feed, and even partial protection could be acceptable and economical in that setting. A plant-based vaccine to protect poultry from Newcastle disease virus was developed by Dow and approved by the U.S. Department of Agriculture in 2006. That was a notable milestone in that
Technologies for Improving Animal Health and Production 199 it was the first plant-based vaccine to win regulatory approval. However, the vaccine is not expressed in whole plants but is produced by means of genetically modified plant cells cultured in steel fermenters; this production method resolves many issues related to containment (Moschini, 2006). One approach to vaccine development uses an RNA virus as a vector, mediated by Agrobacterium tumefaciens to deliver genes that are expressed throughout the recipient plant. The transformation and expression system is efficient and is referred to as a launch vector. The system is not based on the natural mode of virus infection, so there is no size constraint on the gene construct within the vector. Its advantage is that the whole plant biomass can be infused without concern about the vectorâs cell-to-cell movement. The time from inoculation to harvest is 2 to 4 days. A large amount of antigenâup to hundreds of kilogramsâcan be produced in a greenhouse, without the need to grow large batches of crops. Vaccines made this way can be highly suitable for a region where local delivery is important (Yusibov et al., 2002). In addition to tissues, vaccines could be produced and stored as seeds, which would provide a stable form in which the protein will not degrade over time. The choice of the crop would determine how the vaccine is administered: some plants can be consumed raw, but others must be pro- cessed. Processing introduces the potential of heat or pressure treatments to destroy the protein. Cereal crops are attractive for expressing subunit vaccines because they can produce proteins in their seeds that are stable for long storage periods. For animal vaccines, the plant selection could be based on what is eaten as a major part of the diet. The production of vaccines from plants has the following advantages over traditional systems that involve the administration of dead or attenu- ated viruses: plant-based vaccines offer greater biological security because plants do not become contaminated with human or animal pathogens; plant-based vaccines can be administered orally in the form of a single- dose capsule, so the use of needles and syringes is avoided; the vaccines do not have to be kept refrigerated; the production system is economi- cal and can easily be put into large-scale production with conventional agricultural techniques; and the system offers the possibility of producing multi-component vaccines (Agrobiotechnology Institute, http://www.agro- biotecnologia.es/en/index.htm). A disadvantage is the potential variation in the concentration of vaccine produced in different plants, which might make it difficult to feed an efficacious dose. DNA Vaccines DNA vaccination stimulates the immune response by introducing into the host naked DNA that codes for antigens of a pathogen. The protein synthesis machinery of the host cell expresses the antigen and stimulates a
200 Emerging Technologies to Benefit Farmers specific response by the hostâs immune system. In theory, DNA vaccines can be manufactured far more easily and less expensively than vaccines com- posed of inactivated pathogens, protein subunits, or recombinant proteins. Other potential advantages include stability, resistance to extreme tempera- tures, efficacy as an oral vaccine, and the ability to introduce multiple an- tigens (Mwangi et al., 2007). However, substantial development is needed before DNA vaccines become an alternative to conventional methods. Most of the experimental DNA vaccines have not shown as great protec- tive immunity as conventional vaccines, but new technologies, such as the coating of colloidal gold with DNA, that are in development could improve effectiveness. If future research can deliver a DNA vaccine that offers pro- tective immunization, this approach would add flexibility to the custom designing of vaccines for regional needs. For instance, it is easier to change the sequence of an antigenic protein or to add heterologous epitopes. The protective immunity of the expressed protein can be easily evaluated after the DNA is injected into a model animal, such as the mouse. This simple, elegant method could quickly allow researchers to learn about the effec- tiveness of candidate antigens. The final goal of effective DNA vaccines is considered to be far in the future because of the many unresolved problems, but the potential high payoff will continue to draw investment. Animal Disease Surveillance It is pointless to develop and deliver drugs and vaccines without know- ing which syndromes are present in a region, because protecting an animal against one pathogen only to have it succumb to another will not reduce the burden of disease on a small-holder farmer. Developing a database of such information will require field research, trained technicians, and diag- nostics. The relatively new World Animal Health Information Database managed by the World Organization for Animal Health (OIE) is a signifi- cant database that tracks disease prevalence in all regions of the world. In cooperation with the Food and Agriculture Organization of the United Nations (FAO), the OIE is investigating disease rumors that surface on ProMED or other non-scientific sources of information; these early warning systems serve as good alert systems for emerging disease outbreaks. The use of satellite-based remote sensing technologies could be useful as early warning systems for the emergence of serious infectious diseases, particularly those that are transmitted by arthropods. The FAOâs Emer- gency Prevention System (EMPRES) for Transboundary Animal and Plant Pests and Diseases program currently uses remote sensing technologies to determine the Normalized Difference Vegetation Index (NDVI), and the use of such data has led to the successful advanced prediction of Rift Val- ley fever outbreaks (FAO, 2008). Similar technologies have been used for
Technologies for Improving Animal Health and Production 201 the advanced notification of blooms of desert locus and of outbreaks of Venezuelan Equine Encephalomyelitis (FAO, 2008). Inexpensive diagnostic tests, like that developed for rinderpest (Yilma, 1989; Ismall et al., 1994), are needed for disease detection and vaccination campaigns. Other similar rapid pen-side tests for the recognition of infec- tious diseases have been developed and are in use, such as the field diagnosis of human and avian influenza outbreaks. Increasing in greater numbers are the development, validation, and deployment of rapid RT-PCR technologies for accurate diagnosis of a variety of diseases affecting SSA and SA. These tests require only a nasal swab as a sample and are not sensitive to the effect of higher temperatures in the transportation to diagnostic laboratories. Furthermore, emerging technologies, such as biosensors (Box 6-8), are promising because of their sensitivity, speed, portability, and ease of use and could be developed for a variety of surveillance efforts and especially useful in resource-constrained countries in SSA and SA. Moreover, if farmers have BOX 6-8 Biosensors for Rapid Diagnosis A biosensor is an electronic device that contains a biological receptor close to a transducer that converts the interaction between the receptor and the target of analysis (such as a pathogen) into a measurable electric signal whose strength is related to the concentration of the target. There are a number of experimental configurations and platforms. In one type of biosensor, very thin nanowires are bound to a biomolecule, such as a short piece of DNA (an oligonucleotide), whose conformation changes when a target binds to it; the change in confor- mation produces a change in charge that is detected by and transmitted by the nanowire. Biosensor technology has progressed quickly in recent years because of the homeland security interest in rapid detection of small amounts of biologi- cal agents that could be used for terrorism. Several technologies feed into the development of biosensors, including genomics, nano- and micro-fabrication and instrumentation, chemical and polymer science, and signal processing and data transmission. New generations of biosensors have automated signal transmission to record and send information from remote locations. The key advantages of biosensors are sensitivity, speed (4 to 6 minutes vs. 2 hours for the polymerase chain reaction), portability, and ease of use. Specificity, cost, and manufacture will need additional research. SOURCE: Evangelyn Alocilja, Michigan State University, presentation to committee, August 17, 2007.
202 Emerging Technologies to Benefit Farmers tools to detect the presence of disease, they are more likely to seek out a drug or vaccine. Farmersâ confidence in medical treatment and vaccination depends on their seeing a benefit, which they will not if a problem is not solved by a drug or vaccine that targets a single pathogen (Guy Palmer, Washington State University, presentation to committee, September 24, 2007). Transgenic Arthropods The genetic engineering of arthropods to alter vector competency and disease transmission could conceivably reduce vector-borne diseases in animals, plants, and humans. By genetically manipulating vectors, such as mosquitoes, and eventually changing their life-cycle dynamics in the field, the ability of local populations of arthropod vectors to transmit diseases could be significantly altered (Scott et al., 2008). Needs for drug and Vaccine Development for sub-saharan africa and South asia Knowledge of Pathogen and Host Variability In addition to the very presence of a pathogen, pathogen serotype is important in drug and vaccine development. For example, although it is not difficult to find conventional vaccines for many major animal diseases, it is not clear that vaccines based on pathogen serotypes in the industrialized world would necessarily provide protection to animals in SSA and SA, be- cause a given causative agent might have different immunogenic character- istics in different regions. Moreover, most vaccines have not been tested on the indigenous animals to be protected, and knowledge of the diversity of the major histocompatibilty complex in a region must be accounted for. Genomic tools can be used to identify differences in geographic strains of a pathogen by comparing highly useful epitopes (that offer immune protection for the host) according to the homology of a pathogen in two distinct regions of the world. Sequencing can help to identify potential antigens of the pathogen of interest that could be evaluated as vaccines. If a pathogen has a standard reference sequence, partial sequencing can help to identify differences in epitopes of a similar strain in a developing country. Faults in a vaccine could be identified and result in the design of a better vaccine for a region. Genomics research on important animal pathogens should be supported because it will lead to better vaccine designs (Dertzbaugh, 1998).
Technologies for Improving Animal Health and Production 203 Adjuvants A vaccine stimulates a hostâs production of antibodies specific to anti- gens of the pathogen. For various reasons, however, vaccines do not always produce an immune reaction strong enough to protect the host. That is especially true of parasitic diseases that require a vaccine to elicit strong T-cell-mediated immunity in addition to stimulating protective antibodies. Adjuvants are compounds added to vaccines that cause the immune system to respond more vigorously, and they include organic and inorganic salts, virosomes, and experimental compounds. Most adjuvants have been devel- oped by pharmaceutical companies and held as proprietary property (Guy Palmer, Washington State University, presentation to committee, September 24, 2007). There is a need to develop and make available adjuvants to improve current vaccines. Distinguishing Vaccination from Infection Livestock and meat from regions where infectious diseases persist are prohibited from exportation to other countries regardless of whether the animals have been vaccinated. Until recently, it was not possible to dis- tinguish between vaccinated and diseased animals in that both will have produced antibodies to a pathogen. That has served as a major barrier to entry markets for farmers in SSA and SA where several diseases persist. The ability to distinguish between animals exposed to a whole virus and vaccinated animals consistently and reliably would be important in the development of vaccines. Such a diagnostic system for differentiating infected from vaccinated individuals (DIVA) already exists and has been applied successfully for pseudorabies and avian influenza (Pasick, 2004). In addition, several DIVA vaccines and their companion diagnostic tests are on the market and can be applied for foot-in-mouth disease and classical swine fever (Pasick, 2004). Attenuated vaccines have been widely used in SSA and SA for the control of diseases such as peste des petits ruminants, sheep and goat pox, and hemorrhagic septicemia. The use of preimmunization, or the deliberate infection of animals with viable pathogenic organisms followed by a treatment with chemotherapeutic agents, for several homoparastic dis- eases such as Anaplasma marginale, Babesia bovis, Ehrlichia ruminantium, and Theileria annulata in SSA and SA are not safe technologies because they do not propagate the infectious organisms to naÃ¯ve populations. In this respect, live attenuated vaccines provide better immunity than subunit or killed vaccines. The development of stable strains and insertion of marker genes into these strains to differentiate them from wild-type strains would facilitate vaccine deployment for diseases most relevant to SSA and SA.
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