May 2005

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Douglas D. Bannerman1 and Robert J. Wall2

Mastitis is an inflammation of the mammary gland that most frequently develops in cows in response to intramammary bacterial infection. Mastitis remains one of the most prevalent and costly production diseases in dairy herds worldwide. In the U.S., economic losses associated with mastitis are estimated to approach $2 billion annually. These losses are primarily attributable to decreased milk production, however, other contributing factors include veterinary costs, replacement costs for culled animals, and loss of premium payments due to increased milk somatic cell counts. Although management practices can reduce the incidence of mastitis, the ubiquitous nature of mastitis pathogens in the cow's environment precludes complete prevention through good management alone. Selective breeding, which has the potential to enhance certain traits associated with production, is of limited use in preventing mastitis due to the low heritability of the disease. Thus, under current conditions intramammary infections and the development of mastitis remain inevitable. Antimicrobial therapeutics for treating mastitis remain limited and are often sub-optimal. The development of new strategies for the prevention and/or treatment of mastitis should continue to be a high priority among animal health initiatives.

Staphylococcus aureus-induced mastitis
Staphylococcus aureus
are one of the most prevalent bacteria that cause mastitis and are responsible for ~25-30% of all intramammary infections1. Mastitis caused by S. aureus is most often subclinical, however, a substantial incidence rate of clinical mastitis is associated with this pathogen. S. aureus is regarded as a contagious mastitis pathogen because it is commonly spread from infected to non-infected cows at milking2. Although the main reservoir of these bacteria is infected udders, S. aureus have been recovered from surfaces all over the cow and can be readily disseminated from animal caretakers who are carriers of this pathogen. Following penetration of the teat canal, these bacteria release a variety of toxins and products that are injurious to the milk-producing cells of the mammary gland and impair the gland's immune defense mechanisms. The intramammary formation of abscesses around these bacteria and their capacity to reside intracellularly contribute to the ability of S. aureus to establish a chronic infection that can persist for the life of the animal1. The result of establishment of a subclinical, chronic intramammary infection is long-term decreased milk production that often goes undetected.

Effective strategies for the prevention and treatment of mastitis caused by S. aureus remain elusive. Vaccines to prevent the establishment of intramammary infection by this pathogen have been around for decades, however, their efficacy has been limited. Their limited effectiveness may be due, in part, to improper immunization schedules, ineffective adjuvant formulation, and their inability to cross-protect against various strains. Since multiple strains can be present within any one herd or even within an individual cow3, vaccines or other strategies with less restrictive strain specificity will be required to decrease the incidence of mastitis caused by S. aureus.

Once established, S. aureus infections are difficult to treat. These bacteria are able to reside intracellularly and are shed periodically into the milk. Their residence inside of the cells within the gland and the formation of abscesses around foci of infection restrict their contact with administered antibiotics. The percentage of S. aureus-infected animals that can be cured during lactation with currently approved antibiotics is only between 10 – 30%. In a recent study of the efficacy of pirlimycin, a common antibiotic used in the treatment of these infections, only 13% of S. aureus intramammary infections were cured following the recommended two-day therapy4. Extending the therapy to five consecutive days increased the cure rate to only 31%, a level that is below the break-even point at which costs incurred through treatment are balanced by increased production and the premium pay associated with milk containing lower numbers of milk somatic cells2. Thus, treatment options for lactating cows infected with S. aureus remain suboptimal.

Lysostaphin treatment of S. aureus intramammary infections
Lysostaphin is a proteolytic enzyme produced by Staphylococcus simulans that cleaves specific bonds found in the peptidoglycan cell wall component of S. aureus. Due to its potent lytic activity towards this pathogen, lysostaphin's ability to kill S. aureus has been evaluated in several animal disease models. In a model of endocarditis in rabbits, lysostaphin was more effective than vancomycin at reducing valve vegetation bacterial counts5. Further, lysostaphin has been reported to be effective in the treatment of eye infections caused by S. aureus6. With the advent of antibiotic-resistant bacteria, another key finding of lysostaphin's ability to kill S. aureus is its equivalent lytic activity toward both methicillin-resistant and sensitive strains7.

The efficacy of lysostaphin for the treatment of S. aureus-induced mastitis has also been evaluated in a variety of animal models, including mice, goats, and cows. Infusion of lysostaphin into the S. aureus-infected glands of mice was shown to significantly reduce the number of viable bacteria8. Lysostaphin has also been demonstrated to have a cure rate of ~20% when used to treat S. aureus intramammary infections in lactating cows9. Although this rate is comparable to commonly used antibiotics, lysostaphin's targeted specificity and low toxicity may make its use more advantageous. Further studies evaluating formulation, dosing, and treatment durations will be needed to determine whether its efficacy as an intramammary infusate can be enhanced.

A novel strategy for prevention of S. aureus-induced mastitis
Novel research with transgenic animals has revealed an efficacious strategy for the use of lysostaphin in the prevention of mastitis caused by S. aureus. Initial research in this area was conducted using a mouse model of mastitis. Researchers established that the engineering of mice to express and secrete lysostaphin into milk conferred resistance against S. aureus intramammary infection10. The protection conferred was dependent upon the level of expression with the highest expressing line demonstrating complete resistance. After establishing the potential of this strategy in a mouse model of mastitis, the next logical step was to test this proof of concept in the actual economically relevant animal, the dairy cow.

In the first study of its kind to explore the potential of genetic engineering to enhance disease resistance in cattle, researchers from the USDA's Agricultural Research Service developed transgenic cattle that expressed lysostaphin in the mammary gland11. Using a system that restricted expression to the mammary secretory epithelium, the scientists were able to develop cows that secreted lysostaphin directly into the milk of the gland. Milk levels of lysostaphin varied between the transgenic animals and ranged from 0.9 to 14 μg/ml. Of the mammary glands of three transgenic cows that were infused with S. aureus, only 14% became infected. In comparison, 71% of the quarters challenged in the control animals became infected. Even the transgenic cow expressing the least amount of lysostaphin (i.e., 0.9 μg/ml) had an infection rate of only 33%. The highest expressing cow of the three transgenic cows infused with S. aureus, who expressed 11 μg/ml of lysostaphin in her milk, was completely resistant to infection. The concentration of lysostaphin in the milk of the transgenic cows remained fairly consistent throughout lactation. The consistent level of expression conferred resistance throughout lactation as the highest expressing cow maintained complete resistance to multiple challenges throughout this period.

The data presented by these researchers suggest that lysostaphin expression in the gland prevents the establishment of infection. The authors monitored cows for both a febrile response and for the induction of acute phase protein synthesis, the latter of which is a sensitive marker of infection. In addition to these systemic indicators, individual quarters were monitored for changes in milk somatic cell counts, which during mastitis are primarily composed of white blood cells that play a role in combating infection. As expected, control cows that developed established S. aureus infections had increased body temperatures, elevated levels of circulating acute phase proteins, and increased milk somatic cells counts. In contrast, the transgenic animals demonstrated none of these signs of inflammation. These data suggest that lysostaphin prevents infection through its bactericidal properties and, thus, prevents the onset of inflammation.

Transgenic expression of antibiotic appears to have great promise when compared with traditional antibiotic therapy or intramammary infusion of lysostaphin. The key difference between these approaches is that one prevents the establishment of infection, whereas, the other is used to cure already established infections. It may be that once an S. aureus infection becomes established, it may be too late to intervene. Thus, prevention of infection, perhaps using a transgenic approach as demonstrated by these researchers, may be the more effective way to address this mastitis problem.

Clearly there are public concerns regarding the use of transgenic animals for food production. However, the approach demonstrated by the authors had little effect on milk composition. Further, others have demonstrated that lysostaphin has a low immunogenicity, thus, it remains unlikely to generate an allergic response if consumed12. Finally, in an age of considerable concern regarding the development of antibiotic resistance, alternatives that can minimize their use in food-borne animals, such as in the treatment of mastitis, require further investigation.

1. Sutra L & Poutrel B (1994) Virulence factors involved in the pathogenesis of bovine intramammary infections due to Staphylococcus aureus. J Med Microbiol 40, 79-89

2. Sears PM & McCarthy KK (2003) Management and treatment of Staphylococcal mastitis. Vet Clin North Am Food Anim Pract 19, 171-185, vii

3. Kerro Dego O, van Dijk JE & Nederbragt H (2002) Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion. A review. Vet Q 24, 181-198

4. Gillespie BE, Moorehead H, Lunn P, Dowlen HH, Johnson DL, Lamar KC, Lewis MJ, Ivey SJ, Hallberg JW, Chester ST & Oliver SP (2002) Efficacy of extended pirlimycin hydrochloride therapy for treatment of environmental Streptococcus spp and Staphylococcus aureus intramammary infections in lactating dairy cows. Vet Ther 3, 373-380

5. Climo MW, Patron RL, Goldstein BP & Archer GL (1998) Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis. Antimicrob Agents Chemother 42, 1355-1360

6. Dajcs JJ, Thibodeaux BA, Hume EB, Zheng X, Sloop GD & O'Callaghan RJ (2001) Lysostaphin is effective in treating methicillin-resistant Staphylococcus aureus endophthalmitis in the rabbit. Curr Eye Res 22, 451-457

7. Huber MM & Huber TW (1989) Susceptibility of methicillin-resistant Staphylococcus aureus to lysostaphin. J Clin Microbiol 27, 1122-1124

8. Bramley AJ & Foster R (1990) Effects of lysostaphin on Staphylococcus aureus infections of the mouse mammary gland. Res Vet Sci 49, 120-121

9. Oldham ER & Daley MJ (1991) Lysostaphin: Use of a recombinant bactericidal enzyme as a mastitis therapeutic. J Dairy Sci 74, 4175-4182

10. Kerr DE, Plaut K, Bramley AJ, Williamson CM, Lax AJ, Moore K, Wells KD & Wall RJ (2001) Lysostaphin expression in mammary glands confers protection against Staphylococcal infection in transgenic mice. Nat Biotechnol 19, 66-70

11. Wall RJ, Powell AM, Paape MJ, Kerr DE, Bannerman DD, Pursel VG, Wells KD, Talbot N & Hawk HW (2005) Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nat Biotechnol 23, 445-451

12. Daley MJ & Oldham ER (1992) Lysostaphin: Immunogenicity of locally administered recombinant protein used in mastitis therapy. Vet Immunol Immunopathol 31, 301-312

Douglas D. Bannerman1 and Robert J. Wall2
Bovine Functional Genomics Laboratory
2Biotechnology and Germplasm Laboratory
Agricultural Research Service, U.S. Dept. of Agriculture
Beltsville, MD

César Petri and Lorenzo Burgos

Conventional breeding of temperate fruit trees is constrained by their extensive reproductive cycle with long juvenile periods, complex reproductive biology, and high degree of heterozygosity. As the commercial production of transgenic annual crops becomes a reality in many parts of the world, the question remains whether genetically engineering fruit trees will find commercial application.

Gene transfer for fruit tree improvement has several inherent advantages. Once a useful transformant is isolated, vegetative propagation, which is the normal method of multiplying fruit trees, provides unlimited production of the desired transgenic line. Fixation through the sexual cycle is unnecessary and inconvenient if commercially-accepted cultivars are transformed. Since production of most fruit tree species is based on a few cultivars, the impact of transforming one of them would be significant. Currently, however, the only transgenic fruit tree commercially produced is papaya (Carica papaya L.) resistant to PRSV (papaya ringspot virus). (Further information on commercialized transgenic crops can be found at

Transformation methods
Microprojectile bombardment has been used to transform papaya1. The DNA most commonly transferred to fruit trees is from disarmed and genetically engineered Agrobacterium strains, which drive foreign DNA into plant cells. Together with the gene of interest, the genes required for transformation are transferred, including marker genes that allow selection of transformed cells. Among the most commonly used selection genes are the neomycin phosphotransferase gene (nptII), which confers resistance to aminoglycoside antibiotics, and the phosphinothricin acetyl transferase gene, that confers resistance to the herbicide phosphinothricin (see review2). However, given public concern with the introduction of antibiotic resistance genes into food, methods to eliminate selection genes from the transformed plants and strategies that avoid selection of transformed cells with antibiotics are being developed. However, these new methodologies have yet to be applied to the production of transformed fruit trees.

Recent Advances
Much work has been published reporting improved methods using only marker genes and integration of putative beneficial genes, but without sufficient evaluation of the effect on the transformed plants. Additionally, some advances have also been reported specifically in the transformation of fruit trees (see review3 for more details).

o Fire blight and scab resistant apples and pears have been produced by integration of different genes, and plant growth has been modified by introducing the rol B gene in apple rootstocks. Additionally, transgenic apple releases to test possible resistance to pathogenic fungi and bacteria (B/NL/02/03/ and B/DE/03/140) or the effect of cDNA from apple self-(in)compatibility alleles on pollen-pistil incompatibility (B/BE/03/V1) are included in the European Commission data base (

o Introduction of genes of interest subsequent to transformation of apricot seeds and plum hypocotyls has produced plants with increased sharka virus resistance.

o Cherry rootstocks with improved rooting and Basta herbicide resistance were obtained after transforming shoots with A. rhizogenes, and peaches with increased branching and reduced rooting were engineered using a "shooty mutant," A. tumefaciens, strain.

o Different grape cultivars have been transformed with genes coding for: 1) chitinases to confer resistance to pathogenic fungi; 2) different virus coat proteins; and 3) peptides with antimicrobial activity. Grape cultivars have also been transformed with the gene DefH9-iaaM, which induces parthenocarpy in flowers of different species.

o Japanese persimmons and walnuts transformed with the cryIA(c) gene were more resistant to lepidopteran pests, and kiwifruit transformed with the rol A, B and C genes had an improved rooting ability.

o Major objectives of Citrus transformation have been resistance to citrus tristeza virus (CTV) mediated by pathogen-derived genes, resistance to Phytophthora citrophthora using antifungal proteins, and tolerance to salinity by introducing HAL2 yeast-derived genes. In addition, Arabidopsis floral genes, such as LEAFY (LFY) or APETALA1 (AP1), constitutively expressed in citrus seedlings from apomictic seeds, shortened the juvenile phase and promoted precocious flowering. Transgenic plants produced normal, fertile flowers that set fruits containing seeds. These traits were transmitted to the progeny, resulting in trees with a generation time of one year from seed to seed. Whereas LFY lines showed alterations in growth and development, AP1 plants were adult and fully normal. Citrus plants expressing bovine lysozyme and snowdrop lectin are being evaluated in the greenhouse and in the field for their resistance to citrus canker (Xanthomonas axonopodis pv. citri) and insects, respectively.

Transformation Constraints
Currently, transformed seedlings from the cultivars 'Rio Red', expressing lectin, and 'Carrizo', expressing lysozyme, are currently the only Citrus field tests in the USA. Citrus cultivars have very long juvenile periods, and transformation of adult material would be preferable. Transformation of adult tissues of 'Pineapple' sweet orange produced plants that flower and set fruits in 14 months, whereas sweet oranges (which account for approximately 70% of world citrus production) need up to 20 years to completely lose juvenile characteristics and commence production. However, transformation efficiencies in adult Citrus are much lower than in juveniles. For instance, transformation efficiency in adult sweet orange 'Pineapple' is one-third of that obtained with apomictic seedlings of this cultivar. Similarly, juvenile material of Prunus is more easily transformed and, in most cases, has been transformed only with marker genes in preliminary studies. In all cases of integration of "genes of interest" in Prunus, hypocotyls have been used for transformation.

Genotype is a major determinant for transformation, and procedures developed for one cultivar are often not suitable for other cultivars. This is the most serious hindrance to the application of gene transfer technologies to fruit crops. For species with cultivars that can be reliably transformed, the literature generally reveals that few genotypes of a particular species are being transformed and, in some cases, that these genotypes are not commercially important.

Meristem transformation may eliminate the need for regeneration in production of transgenic plants, allowing genetic manipulation of established cultivars. However, high explant mortality and difficulties controlling Agrobacterium growth have limited the development of this methodology. Recently, a reliable procedure for transformation of different grape cultivars has been developed4. The authors generated "meristematic bulk" (MB) tissue from in vitro shoots by mechanical (dissection of the apical dome) and chemical (progressive increase in cytokinin concentration) treatments that abolish shoot apical dominance and also promote basal meristem proliferation. The MB tissue is a large aggregate of meristematic tissue with high regenerative competence, which can be transformed efficiently by Agrobacterium given the large number of dividing cells. This system seems to be easily adapted to other fruit trees; for instance, MBs have been produced from three apricot cultivars with similar regeneration efficiencies, according to data obtained in our laboratory. We are presently conducting experiments to transform MB tissue and regenerate transgenic shoots from apricot.

Selection of transformed regenerants is a critical step in any transformation procedure. Most commonly in fruit trees, antibiotics have been used as selection agents after integration of genes that confer antibiotic resistance. Concentration of the selective agent and timing of application must be optimized for each plant species.

Selection of transformed shoots is often complicated by the inactivation of the selection agent by transformed cells and persistence of Agrobacterium in the explants, which permits regeneration of non-transformed shoots (escapes), sometimes at a high frequency.

Despite the economic importance of Prunus, transformation technology is not available for most Prunus species, which may be due to difficulties posed by adventitious regeneration and/or a high sensitivity to antibiotics. Whereas in Citrus, pear, walnut, or olive, selection is provided by 100 mg/L kanamycin, in Prunus inhibitory concentrations are frequently much lower (5 to 10 mg/L in almond, for instance), and specific selection strategies are often necessary.

Future Perspectives
The future of genetic transformation as a tool for breeding fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. Yet another obstacle is that European law will neither allow deliberate release of plants carrying antibiotic resistance genes after 2004 nor their commercialization after 2008 (Directive 2001/18/EEC of the European Parliament and the Council of the European Union). Therefore, development of procedures to avoid the use of antibiotic selection or to allow elimination of marker genes from the transformed plant will be a research priority in the coming years.

1. Fitch MMM, Manshardt RM, Gonsalves D, Slightom JL, Sanford JC (1990) Stable transformation of papaya via microprojectile bombardment. Plant Cell Rep 9,189-194

2. Miki B & McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety, J Biotechnol 107, 193-232

3. Petri C & Burgos L (2005) Transformation of fruit trees. Useful breeding tool or continued future prospect? Transgenic Res 14, 15-26

4. Mezzetti B, Pandolfini T, Navacchi O & Landi L (2002) Genetic transformation of Vitis vinifera via organogenesis. BMC Biotechnology 2, 18

César Petri and Lorenzo Burgos
Departamento de Mejora de Frutales at CEBAS-CSIC
Murcia, Spain

Tayyab Husnain

Genetic engineering of crop plants has emerged as a powerful tool for creating and preserving genetic diversity, which can then be exploited through conventional methods of plant breeding. Many important traits incorporated through genetic engineering techniques, including herbicide resistance, drought and salt tolerance, improved colors in fiber and flower crops, resistance to water logging, nutritional benefits, and longer shelf lives, result from symbiotic relationships between biotechnologists and plant breeders. These relationships have contributed to the successful incorporation of insect resistance in a number of plant species. An important source of insect resistance genes is a gram-positive soil bacterium, Bacillus thuringiensis, encoding insecticidal crystal proteins toxic to a selective range of insects. A number of genetically modified insect resistant crop species have been tested under natural conditions and many are enjoying commercial status. The global area under transgenic crop cultivation for the year 2003 was 44.2 million hectares, and the main transgenic crops were soybean, corn, cotton, and canola1.

Transgenic crops should be extensively studied and field trials conducted to assess possible risks posed by these plants. When conducting risk assessment trials, one should consider the biology of the crop, the introduced trait, the receiving environment, and the interactions among these aspects. Other considerations include the source of the insect resistant gene and the method by which it is introduced into the plant. Target insects characteristics should be studied to help prevent the emergence of pest resistance. The choice of experimental design, considerations about resistant management strategies, and biosafety measures are also important to consider when conducting field trials.

We report the first field trial of two transgenic lines of Indica Basmati rice (B-370) expressing either the cry1Ac or cry2A genes. We grew the transgenic lines under field conditions for two consecutive years, using either a randomized complete block design (RCBD) or a split plot design (2000-2001). Additional lines, simultaneously expressing the two Bt cry1Ac and cry2A genes, were also sown under field conditions using the RCBD (2001), and at two different locations (2002) using the split plot design. A strategy that combines a refugia strategy with the use of transgenic lines containing a high dose of Cry proteins expressed simultaneously from two unique Bt genes is effective in the delay of resistance against Bt toxins2.

Biosafety precautions were taken during all field trials. Sixty neonate larvae of the yellow stem borer (YSB, Scirpophaga incertulas) were artificially introduced onto each plant in three installments (2000 and 2001), while during the second year, plants were infested with three freshly hatched egg masses. Data were collected on dead hearts/leaf damage and whiteheads at the vegetative and flowering stages, respectively. The transgenic Indica rice lines were significantly resistant to the applied target insects (p<0.01). Natural infestations of rice skipper (2000) and rice leaf folder (RLF, Cnaphalochrocus medinalis; 2001 and 2002) were also observed during these trials, and the transgenic rice was statistically superior to the untransformed counterparts in resisting damage by these insects. Transgenic lines displayed up to 100 and 98% resistance against YSB at the vegetative and flowering stages (Fig. 1), respectively, with 98% additional resistance against RLF as compared with the untransformed control.

Figure 1. Basmati and control rice showing resistance to Yellow Stem Borer
A: Transgenic Line (showing 8% white heads)
B: Untransformed control (showing 48% white heads)

The presence of cry genes was confirmed using Dot blot, PCR, and Southern blot analysis, while ELISA and Western bolt analysis confirmed the expression of Cry proteins. All lines expressed higher levels of Cry proteins when compared with commercially released cultivars of Bt cotton, maize, and potato.

Variation in some morphological characteristics, e.g., the average number of tillers, plant height, and maturity, were observed. Lines expressing two genes were superior for average number of tillers, plant height, days to maturity, and resistance to lodging as compared to the lines expressing one gene and the untransformed control. Transgenic lines produced up to 59% more grains than control plants under artificially augmented conditions, while up to an 8% increase was recorded under natural infestation conditions alone.

All transgenic Basmati rice lines expressed a high level of Cry proteins, and although toxin titer substantially decreased with increasing age of the plants, it remained well within the range to kill target insects effectively. Gene expression was constitutive, as toxins were quantified in different parts of the plants, specifically leaves, stem, roots, panicle, seeds, and kernel. Although the promoters were not tissue specific, significant variation was recorded for Bt insecticidal protein quantified in different parts of the plants, perhaps due to a difference in water and protein contents. Some variation in cry expression in different parts of the plants has been reported3,4. In this study, the expression of cry2A was significantly less than cry1Ac, which may be due to the fact that cry2A was under the control of the CaMV 35S promoter–the ubiquitin promoter is reported to be more active than CaMV 35S in transgenic rice plants5.

The transgenic lines had no effect on the presence of non-target insects (insects belonging to orders other than diptera and lepidoptera) nor on the germination of three local varieties of wheat. A 0.18% level of cross pollination was calculated in experimental lines. It was also observed that the transgenic lines released Bt toxins from roots into Murashige and Skoog (MS) basal medium (1.5 ng/ml of medium), hydroponic cultures (1.6 ng/ml of solution) and into the soil in trace amounts, which could be detected through sandwich ELISA.

The insect resistant Basmati rice displayed short plant height and early maturity characteristics. Its nutritional value was comparable to the parent lines. However, transgenic Basmati rise is not currently in a variety development program, likely due to a negative public perception of transgenic organisms. The advantages of developing insect resistant rice include greater yields with less input, avoidance of excess chemical spray, and superior performance during insect pest epidemics. These studies also provide additional data on the biosafety of transgenic crops, acquired insect resistance to Cry proteins, and the fate of transgenes in the soil.

1. James C. (2003) Global status of commercialized transgenic crops. ISAAA Briefs No. 30. Ithaca NY.

2. Cohen MB, Gould F, Bentur JS. (2000) Bt rice: Practical steps to sustainable use. IRRN (25)2, 4-10

3. Husnain T, Jan A, Maqbool SB, Datta SK, Riazuddin S. (2002) Variability in expression of insecticidal Cry1Ab gene in Indica Basmati rice. Euphytica 128, 121-128

4. Bashir K et al. (2004) Field evaluation and risk assessment of transgenic Indica Basmati rice. Mol. Breeding 13, 301-312

5. Koziel MG et al. (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/technology 11, 194-200

Tayyab Husnain
National Centre of Excellence in Molecular Biology
Lahore, Pakistan

Robert Gulden*, S Lerat, M Hart, R Campbell, J Powell, P Pauls, J Klironomos, C Swanton, and J Trevors

The fate of DNA in soil
The introduction of genetically engineered (GE) crops has resulted in the need to address concerns that were of considerably lower significance in strictly conventional crop production systems. They include concerns over food safety, environmental safety, and biodiversity in agroecosystems. In Ontario, Canada, for example, where corn–soybean crop rotations are common, the most abundant GE varieties of these crops, i.e., those resistant to the herbicide glyphosate, comprise about 12% and 55% of the annual acreage of these crops, respectively1 in 2004.

The body of research examining transgenic DNA in the food supply and in the ecosystem has grown substantially in recent years. In the environment, transgene escape through intra- and interspecific hybridization during all stages of field production has been observed2. Although we are beginning to understand the extent of transgenic DNA above the soil surface, very little is known about the fate of transgenic plant DNA in the soil ecosystem. Technical challenges of working in the soil environment, including high spatial variability and difficulties of extracting DNA without co-extraction of polymerase chain reaction (PCR) inhibitors, have been significant limitations to this area of research. However, recent advances in DNA extraction methodology and molecular techniques allow for the development of routine high-throughput techniques to examine comprehensively the fate of plant DNA in the soil environment.

Recently, Lerat et al.3 developed molecular tools for the detection of plant DNA in soil for glyphosate tolerant (Roundup Ready®) corn and soybean. In these crops, the CP4 EPSPS gene (5-enolpyruvylshikimate-3-phosphate synthase), which confers resistance to glyphosate, is identical, while other elements of the inserted gene cassettes of RR corn (event NK603)4 and RR soybean (event 40-3-2)5 are similar.

The challenge lies in DNA recovery
Before transgenic plant DNA can be quantified, it must be recovered from soil. A number of DNA extraction protocols applicable to soil exist; however, most are time and labor intensive and therefore are not suitable for routine extraction of high sample numbers required to estimate DNA content accurately. Thus, a high-throughput method for plant target DNA recovery from soil was developed. The method is based on a commercially available soil DNA extraction kit. The UltraClean-htp kit (Mo Bio Laboratories, Solana Beach, CA) uses the 96-well plate format for high sample throughput and is designed to extract the total soil DNA content from 0.25 to 1.0 gm soil per sample. DNA is extracted with a direct extraction method based on physical and chemical cell disruption and is recovered on a silica membrane. The manufacturer's instructions were modified for maximum plant target DNA recovery. Specifically, glass beads, aurintricarboxylic acid, and AlNH4(SO4)2 were added prior to bead beating. A major limitation of DNA recovery from soil is that PCR inhibitors such as humic and fulvic acids are co-extracted with DNA in many purification protocols. Although humic and fulvic acids are removed by the kit, the addition of AlNH4(SO4)2, a flocculent that precipitates these compounds, improved plant target DNA recovery. Adding one large glass bead to each well also improved plant DNA recovery by increasing the mechanical disruption of cells. DNAses and nucleases are present in soil that can quickly degrade free DNA. The addition of aurintricarboxylic acid, a DNAse and nuclease inhibitor, improved plant DNA recovery as well. The remainder of the DNA extraction method followed the manufacturer's instructions.

Detection of plant transgenes
Specific primers and a molecular beacon for real-time PCR analysis were designed to detect recombinant plant DNA in soil extracts. Detection of the recombinant CP4 EPSPS gene in soil extracts presents a challenge as this gene originated from a strain of Agrobacterium, which is a common soil bacteria. This gene is also present in many other soil bacteria. A second challenge was to discern between the corn and soybean CP4 EPSPS, as the sequences of the recombinant EPSPSs are identical in both species. To overcome these challenges, the junction between the beginning of the EPSPS gene and the adjacent chloroplast transit peptide (CTP) gene was targeted for PCR amplification. The CTP gene adjacent to the CP4 EPSPS is different in corn and soybean and therefore targeting this junction provided the desired species specificity. This approach required only a single probe (molecular beacon), one reverse primer, and two different forward primers. The probe and the reverse primer annealed at the same positions on the CP4 EPSPS gene in corn and soybean. The unique forward primers ensure amplification was initiated either on corn or soybean template DNA, without falsely identifying other EPSPS genes, including those from soil bacteria. Real time PCR was used to quantify plant DNA in soil extracts because this technique is highly specific, extremely sensitive, and can accurately quantify DNA over a large concentration range. In fact, it was possible to detect as few as a single copy of plant target DNA in one microlitre of DNA extract that was added to the PCR reaction. Unfortunately, using a single probe to identify two separate target sequences does not lend itself to multiplexing these PCR reactions. Multiplexing real-time PCR allows for the quantitation of more than one target sequence in the same PCR reaction, but requires separate probes for each target sequence.

What's next?
New molecular techniques and DNA extraction method for recovery of DNA from challenging media have made it possible to develop tools that can be used to routinely detect low quantities of plant DNA in the soil environment. This high-throughput soil DNA extraction method can now be employed to address questions that require analysis of large numbers of samples, including questions such as the persistence and spatial distribution of plant DNA as well as the influence of cropping system on the DNA cycle in the soil. This technique can also be adapted to address questions regarding soil microbial DNA diversity and soil microbe functionality. The introduction of GM crops has provided the impetus, and modern molecular methods and DNA extraction techniques have provided the means, to investigate basic questions about the DNA cycle in the soil environment that have not yet been addressed.

1. Anonymous (2004) Update summer 04. Agcare 14(3), 1-4. [Online]

2. Marvier & Van Acker (2005) Can crop transgenes be kept on a leash? Front. Ecol. Environ. 3, 99-106

3. Lerat et al. (2005) Real-time polymerase chain reaction quantification of the transgenes for Roundup Ready corn and Roundup Ready soybean in soil samples. J. Agric. Food. Chem. 53, 1337-1342

4. See

5. See

Robert Gulden* (corresponding author)
Dept. Plant Agriculture
Univ. of Guelph

Phillip B.C. Jones

In October 1999, Dr. Floyd P. Horn, Administrator of the U.S. Department of Agriculture's Agricultural Research Service, warned a U.S. Senate subcommittee about the vulnerability of American agriculture to a terrorist attack. Other experts have also emphasized the susceptibility of agriculture to a deliberate introduction of animal and plant pathogens at the farm level. Natural barriers that could slow pathogenic dissemination have been thwarted by the concentrated and intensive nature of modern farming practices with highly genetically homogeneous livestock and crops.

Livestock offer an especially attractive target. Terrorists can pick an economically valuable livestock, match the target against a published list of diseases, and select the most accessible pathogen. Many of these organisms are endemic outside the United States and can be isolated from common materials with very little training. And unlike the weapons of bioterrorism, lethal and highly contagious biological agents that affect animals usually do not harm humans.

In fact, experts suggest that the economy, not human health, would experience the greatest impact of an agroterrorism attack. An assault on agriculture would cause direct losses from containment measures and eradication of diseased animals, compensation paid to farmers for destruction of agricultural commodities, and a decrease in international trade as export partners impose protective embargoes.

Following years of warning punctuated by the September 11 terrorist attacks, the federal government has attempted to secure U.S. agriculture. Yet in its March 2005 report, the Government Accountability Office finds that these efforts fall short.

Reshaping Government Agencies to Protect Against Agroterrorism
The legislative and executive branches have altered the roles of federal agencies with the objective of protecting U.S. agriculture from assault. The Homeland Security Act of 2002 established the Department of Homeland Security and charged the department with coordinating efforts to guard against agroterrorism. The Act also transferred most of the USDA's responsibility for conducting agricultural import inspections to DHS. In this way, the DHS should have the capability to recognize and prevent the entry of organisms that might be exploited for agroterrorism. Along with this shift of responsibility for preventing the introduction of plant or animal diseases, the DHS acquired the USDA's authority to inspect cargo manifests, international passengers, baggage and cargo, and to hold suspect articles for quarantine. The DHS also obtained most of the USDA's Plant Protection and Quarantine Unit inspectors.

The Bioterrorism Act of 2002 expanded the duties of the USDA and the Department of Health and Human Services for ensuring agriculture security. The departments gained responsibility for requiring companies, laboratories, and other entities to register materials that could pose a threat to agriculture and human health. The Act also required the USDA and HHS to develop an inventory of potentially dangerous agents and toxins that cause animal, plant, or human diseases. Individuals who possess or use these materials must register with the Secretary of Agriculture or HHS and submit to a background check performed by the U.S. Attorney General.

The Bioterrorism Act of 2002 further authorized the USDA to conduct and support research into the development of an agricultural bioterrorism early warning system. Such a network would enhance coordination between state veterinary diagnostic laboratories, federal and state agricultural research facilities, and public health agencies. To support these efforts, the USDA has the authority to coordinate with the intelligence community for evaluating information about potential threats to U.S. agriculture.

The President issued four directives that further define agencies' responsibilities in protecting agriculture. The directive most relevant to agriculture, Homeland Security Presidential Directive (HSPD)-9, establishes a national policy for defending the country's agriculture and food system against terrorist attacks and major disasters. Under the Directive, DHS serves as the lead agency responsible for ensuring the adequacy of federal, state, and local authorities in responding quickly to a terrorist attack. HSPD-9 also commands the DHS to oversee a national biological surveillance system that will help to differentiate between natural and intentional outbreaks. The Directive tasks the USDA and HHS with developing secure laboratories to enhance diagnostic capabilities for foreign animal and zoonotic diseases. If an agroterrorism attack should occur, then the DHS, USDA, HHS, and Environmental Protection Agency share responsibility for decontamination and stabilization of agricultural production.

Federal agencies have responded to this surge of responsibility and shifting authority. The FDA and USDA have been conducting vulnerability assessments to identify agricultural products most susceptible to terrorist attacks. The USDA and HHS have been forming laboratory networks to enhance diagnostic and monitoring capability. The USDA has established a committee to guide the development of a National Veterinary Stockpile. And the USDA created sixteen Area and Regional Emergency Coordinator positions to help states develop individual emergency response plans.

The GAO found serious shortcomings in these efforts.

Glitches in the System
According to the GAO, the United States still faces complex challenges that limit the ability to quickly and effectively respond to a widespread attack on livestock and poultry. One deficiency lies in the capability to detect such an attack. Many U.S. veterinarians lack the training required to recognize the signs of foreign animal diseases. Current regulations do not even require training in foreign animal disease for those most likely to be called upon if livestock were attacked: USDA-accredited veterinarians. The USDA has been considering a rule that would compel training in foreign animal diseases for accreditation, but the proposed requirement has been residing on a back burner for several years.

The GAO also expressed concern that the USDA uses diagnostic tests to characterize an outbreak at selected laboratories, not at the site of the outbreak. Experts consider on-site testing critical for speeding diagnosis, containing the disease, and minimizing the number of animals that must be slaughtered.

HSPD-9 requires federal and state agencies to develop a National Veterinary Stockpile that contains sufficient amounts of animal vaccines and other therapeutic products for responding to the most damaging animal diseases affecting human health and the economy. And the Directive demands that these therapeutics should be available within 24 hours of an outbreak. Yet the GAO found that vaccine supplies are limited; the USDA usually prefers to immediately slaughter diseased animals rather than vaccinate. The agency maintains vaccines for only one foreign animal infection: foot and mouth disease. Even these vaccines cannot be rapidly deployed, because they would first need to be sent to the United Kingdom for bottling and testing.

The GAO also uncovered several management problems that reduce the effectiveness of routine efforts to protect against agroterrorism. Following the transfer of most USDA agricultural inspectors to DHS, agricultural inspections at ports of entry decreased, even though imports increased. DHS points to a large number of unfilled vacancies for agricultural inspectors, and plans to hire more than 500 inspectors by fiscal year 2006. Others have noted difficulties faced by former APHIS inspectors in the culture of the U.S. Customs and Border Protection service, as well as a lack of clarity about responsibilities shared by the USDA and Customs at U.S. ports.

The GAO's report suggests many changes that federal agencies could implement to address its concerns. However, the organization highlights two recommendations: the USDA should examine the costs and benefits of developing stockpiles of ready-to-use vaccines, and the DHS and the USDA should investigate the reasons for declining agricultural inspections.

Reinvention, not Reshaping
Experts have voiced an overarching concern about the current system for ensuring the security of agriculture and the food supply—the dispersion of responsibility among the many federal and local organizations. This diffuse structure breeds the types of communication problems that the GAO discovered among federal agencies and between the federal and state governments.

In 2004, Peter Chalk of the Rand Corporation advised that safety measures should be standardized and streamlined within the framework of an integrated strategy that cuts across the responsibilities and capabilities of federal, state, and local agencies. "Integration of agriculture and food safety measures," he wrote, "would also serve to reduce jurisdictional conflicts and eliminate unnecessary duplication of effort."

Selected Sources:
Chalk P. (2002) Hitting America's soft underbelly: The potential threat of deliberate biological attacks against the U.S. agricultural and food industry. Rand National Defense Research Institute

Edmonson RG (2005) What's bugging ag inspectors. Journal of Commerce, page 44 (February 14, 2005)

GAO (2005) Homeland security: Much is being done to protect agriculture from a terrorist attack, but important challenges remain. March 8, 2005. Available at:

Segarra AE (2002) Agroterrorism: Options in Congress. Congressional Research Service. July 17, 2002. Available at:

Phillip B. C. Jones, PhD., J.D.
Spokane, Washington

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