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Cotton is the most important cash crop of Pakistan known as "white gold" (Ahmad, 2007). Pakistan is the fourth largest producer of cotton in the world, third largest exporter of raw cotton, fourth largest consumer of cotton, and the largest exporter of cotton yarn. 1.3 million farmers (out of a total of 5 million) cultivate cotton over 3 million hectares, covering 15 per cent of the cultivable area in the country (Abro et al., 2004). Economy of Pakistan is heavily dependent on the cotton and textile sectors which account for 8.2 percent of the value-added in agriculture and about two percent of Gross Domestic Product (GDP) (Maksvytis and Stakisaitis, 2004).
Textile is the largest industry in Pakistan which totally depends on cotton crop but the yield per unit of cotton is low as compared to the rest of world. Multiple factors are responsible for lower yield such as poor management, contaminant seed or high pest attack etc. It is essential to improve cotton to make economy more progressed. Farmers use pesticides as cotton is a plant that is vulnerable to a wide variety of damaging pests. This factor substantially lowers the crop yields and reduces farmer income, which ultimately affect overall economy of country.
Extensive use of pesticides on cotton cause many environmental hazards. Moreover excessive use of pesticides leads to genotoxicity. Genotoxicity and oxidative stress was observed in workers who formulated organophosphorus pesticides (OP) (Nyha, 1985). They reported association of chronic exposure to OP pesticides with increased activities of catalase, Superoxide-Dismutase (SOD) and glutathione peroxidase in erythrocytes, thus increase in DNA damage that may play part in stimulated antioxidant enzymes and increased DNA damage in the absence of depressed acetylcholinesterase levels. Similar study (Shdniaa et al., 2005) showed the same impact of chronic exposure to OP leading to more oxidative stress among works (history of 1 year; age ranged from 23 to 55) and concluded that erythrocyte AChE activity in pesticide workers could be a good monitoring factor and it could be recommended worldwide in pesticide industries.
As the harmful effects of using chemical insecticides are becoming more evident, the need to look for alternatives has become necessary. Among the existing alternatives biological control is the most favorable choice. For varying levels of suitability diversity and adaptation (Hilder and Boulter, 1999). Biological control methods being practiced successfully include the use of pheromones for trapping or disruption of mating behavior, insect growth regulators that interfere with larval development, parasitoids, fungi, viruses and bacteria, which debilitate or cause death in the infected insects (Way and van Emden, 2000). One of the most successful biological control organisms is a naturally occurring bacterial pathogen, Bacillus thuringiensis (generally Known as "bt"). Formulation based on bt have been used for decades as biological insecticides for agriculture and forestry, as well as vector control against mosquitoes and black flies (Boisvert, 2005). Interest in bt proteins has increasesd during the last two decades because of their unique qualities which are unmached by any conventional insecticide (Whalon and Wingerd, 2003).
Bacillus thuringiensis, a gram-positive entomopathogenic bacterium, produces different kinds of crystal inclusions during sporulation. These crystal inclusions are composed of one or various Cry proteins (also called Î´- endotoxins). Some of these proteins are highly toxic to certain insects. They are harmless to most other organisms, including wildlife and beneficial insects (Schnepf et al., 1998; DeMaagd et al., 2001).
The mode of action of Bacillus thuringiensis Cry1 and related proteins follows after ingestion by the insect, a complex process of multiple steps. These include
Solubilization of the crystal to release the Cry proteins in their protoxin form.
Activation of the protoxins by midgut proteases to their active form.
Binding of the toxin to midgut receptors and
Pore formation in the brush border cell membranes, eventually killing the insect.
Recently, an intermediate step has been suggested to link protoxin activation and binding to specific receptors. This step would include the interaction of not fully-activated toxin with the midgut of insect through specific binding sites involved in the activation and oligomerization of the toxin. Oligomeric forms have been suggested in that model, as responsible for the insertion into the membrane and pore formation (Gomez et al., 2002).
There is a lot of literature on 'gene silencing', in which the transgenes remain in the genome, but are no longer expressed. More serious, from the safety point of view, structural instability is observed. There is a tendency for the transgenic DNA to rearrange or become lost in successive generations. This could change the transgenic line in unpredictable ways and could be health and environmental risk (Wan and Cummins, 2005). Biomonitoring is very necessary for bt varieties time to time to judge the status of the variety. Such exercises can be of benefit to our farmers and economy. Once this type of setup is established, we can evaluate any genetically modified crop by this method. This year government of Pakistan is going to introduce seven varieties of bt cotton. There is a need to launch a certification system to boost up economy of Pakistan, to avoid misuses of resources and to help the farmers to get maximum benefit from genuine varieties. Therefore biomonetring is necessary to check the expression level of Cry 1 Ac at different stages of plant.
The most frequently used method is the amplification of genetically modified organisms (GMO) specific DNA by PCR followed by agarose gel electrophoresis, restriction fragment length analysis, southern blot hybridization or DNA sequencing (Walschus et al., 2002). This approach has been successfully applied on transgenic potato, tomato, herbicide resistant maize, bt-maize, soybean and processed products (Hupfer et al., 1997). However, this requires a well-equipped laboratory and suitable methods to optimize the results, while PCR-based methods are time consuming. Immunoassay provides an alternative mean for the detection of GMOs based on the determination of the protein product of the foreign gene. Polyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.
These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected to the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal's serum. (http://en.wikipedia.org/wiki/Polyclonal_antibodies).
Polyclonal antibodies raised either in rabbits (Tapp et al., 1995; Hori et al., 2000) or goats (Sims et al., 1996) have been used. The present study is useful in further detection and quantification of delta endotoxin isolate from tissues of bt cotton.
Japanese biologist, Shigetane Ishiwatari was investigating the cause of the sotto disease (sudden-collapse disease) that was killing large populations of silkworms when he first isolated the bacteriumÂ Bacillus thuringiensisÂ (bt) as the cause of the disease in 1901.
Ernst Berliner isolated a bacteria that had killed a Mediterranean flour moth in 1911, and rediscoveredÂ Bt. He named itÂ Bacillus thuringiensis, after the German town Thuringia where the moth was found. Ishiwatari had named the bacteriumÂ Bacillus sottoÂ in 1901 but the name was later ruled invalid. In 1915, Berliner reported the existance of a crystal withinÂ Bt, but the activity of this crystal was not discovered until much later (ZakharyanÂ et al.,Â 1979).
The entomopathogenic bacterium Bacillus thuringiensis (bt) and its toxins are extensively used for pest control purposes in agriculture, forestry and public health programmes since the 1930. In addition to spray formulations, transgenic plants containing bt genes for the expression of the toxins (bt plants) are commercially available since the mid 1990s and are grown on an increasing percentage of the global agricultural area. A main reason for the importance of Bt as a pesticide is the assumed environmental safety concluded from the high specificity of its endotoxins (Cry proteins) towards a limited number of target organisms, mostly distinct groups of pest insects ( Hilbeck et al., 2006).
Insecticidal crystal proteins are useful agricultural tools. The Cry gene encoding these toxins is a key source of genes for transgenic expression to provide pest resistance in plants. So far, 415 toxins including 181 holotypes have been designated. The current nomenclature of insecticidal crystal proteins is based on amino acid identity and ranks structurally-related toxins together. Each toxin is assigned a unique name incorporating four ranks that clearly indicates amino acid sequence similarity (Shu et al., 2009).
Insecticidal crystal proteins are predominantly comprised of one or more proteins (Cry and Cyt toxins). Cry proteins exhibit experimentally verifiable toxic effects to a target organism or have significant sequence similarity to a known Cry protein. Therefore, bt is a viable alternative for the control of insect pests in agriculture and of important human disease vectors (BravoÂ et al., 2007).
The mode of action of Cry toxins has been characterized principally in lepidopteran insects. As mentioned previously, it is widely accepted that the primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming pores in the apical microvilli membrane of the cells. Nevertheless, it has been recently suggested that toxicity could be related to G-protein mediated apoptosis following receptor binding. Cry proteins pass from crystal inclusion protoxins into membrane-inserted oligomers that cause ion leakage and cell lysis. Subsequently cell lysis and disruption of the midgut epithelium releases the cell contents providing spores a germinating medium leading to a severe septicemia and insect death (Schnepf et al., 1998).
One interesting feature of Cry toxin activation is the processing of the N-terminal end of the toxins. The 3-dimensional structure of Cry2Aa protoxin showed that two Î±-helices of the N-terminal region include a region of the toxin involved in the interaction with the receptor. Also, it was found that a Cry1Ac mutant that retained the N-terminus end after trypsin treatment binds nonspecifically toÂ M. sextaÂ membranes and was unable to form pores onÂ M. sextaÂ brush border membrane vesicles (BBMV). Therefore, processing of the N-terminal end of Cry protoxins may unmask a domain II hydrophobic patch involved in toxin-receptor or toxin-membrane interaction (Bravo et al., 2007).
Polyclonal antibodies and monoclonal antibodies are indispensable tools in the laboratory. Polyclonal antibody production in rabbits is generally associated with multiple injections of antigens and adjuvants and repeated blood sampling procedures.
TheÂ Western blotÂ is anÂ analytical techniqueÂ used to detect specificÂ proteinsÂ in a given sample of tissue homogenate or extract. It usesÂ gel electrophoresisÂ to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typicallyÂ nitrocelluloseÂ or PVDF), where they are probed (detected) usingÂ antibodiesÂ specific to the target protein (Towbin et al., 1979; Renart et at., 1979).
There are now many reagent companies that specialize in providing antibodies (both monoclonalÂ andÂ polyclonalÂ antibodies) against thousands of different proteins.Â These antibodies are used for different research purposes to detect any specific protein, to determine the gene expression level and transgenic conformation.
MATERIALS AND METHODS:
Bacillus thuringiensis grown in laboratory:
Bacillus thuringiensis (CEMB no 30023 strain no PR 17.2) will be grown in 9 cm diameter Petri dishes on LB media by incubating at 370 C for 24 hours. Nutrient media will be autoclaved for 20 minutes at 1210 C at 15 Psi.
Separation of Delta endotoxin:
Spores, parasporal crystal bodies and cell debris will be pelleted from the growth medium by centrifugation for 10 min at 12,000 rpm and washed twice each in 1 M NaCl and in distilled water. The parasporal bodies were separated from the spores by suspending, washing and pelleting the particles three times in cold water, as described by Ibarra and Federici (1986). The parasporal bodies will be suspended in 50 mM NaOH (pH 12.3) and incubated two hours at room temperature. Solubilized delta endotoxins will be recovered in the supernatant after centrifugation for 10 min at 12,000 rpm (Zouari and Jaoua, 1997).
Delta endotoxin detection:
Delta endotoxin detection will be done through Slide agglutination method using specific known antiserum.
Antigen Preparation and inoculation in groups of rabbits:
Two groups of rabbits will be made of 3 pairs each, group A and B. Group A will be given 0.5 mg protein in 1ml and group B 1 mg protein in 1ml in each dose. Antigen used to inject into the rabbits is prepared by the individual labs that are using the rabbits. The antigen will be filter sterilized to reduce the amount of inflammation at the site. Antigen preparations include the use of adjuvants - Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFCA), and Alum - to aid in the stimulation of the immune response. Complete Freund's Adjuvant will be used in the first injection only. The FCA and ICFA are matched in volume to the antigen, making a 1:1 mix. This mix must be thoroughly emulsified. Only ICFA can be used for booster immunizations if FCA was used for the initial immunization. All antigen preparations will be labeled with the complete name of the antigen and the number of the rabbit that is to be injected.
Fractious rabbits may be tranquilized with IM acepromazine (0.1-2.0 mg/kg). Rabbits will be placed in a cat-type restraint bag and transported to the procedure area. The area to be injected will be shaved (6-8" long strip along the back extending 3" on each side of the spine). The area of injection will be wet with water, alcohol or disinfectant solution and wipe with a clean paper towel to remove hair and debris. Injections of the antigen will be given in multiple sites to stimulate the best immune response. A 22 g needle will be used for injections. The rabbits will be re-immunized (boosted) at 21 day intervals until peak antibody titers are reached. ICFA will be used again at 1:1 ratio with antigen. Re-immunization injection sites will be on the opposite side of the back from the initial immunizations (Chu et al., 1979).
A. Ear Bleeds
Administer butrophanol (10 mg/ml) and acepromazine (10 mg/ml) will be mixed at a 1:1 ratio and dose at 0.1 ml/kg. Rabbit will place in a restraint bag. 20gx 1" short beveled needle will be inserted with an appropriate size syringe (10cc usually) into the artery to obtain some blood. Then needle will removed and direct pressure will applied to the entry site until bleeding has completely stopped (Diano et al., 1987).
B. Cardiac Blood Sampling Procedure
Ketamine (dissociative drug; 100 mg/ml) and Acepromazine (tranquilizer; 10 mg/ml) will be mixed at 10:1 ratio and dose at 0.35cc/10 lb. Rabbit will be placed on its back on restraint board. 18g x 1 Â½" needle will be inserted between the 4th and 5th rib on the rabbit's left side at the point where the heart beat is the strongest. Needle will inserted slowly until a flash of blood is observed in the hub, then draw back on the plunger. Alternatively, the needle will be inserted immediately to the rabbit's left of the xiphoid and directed cranially and toward the rabbit's right at a 30 to 45o angle into the heart. After filling the syringe, it will be removed from the needle, leaving the needle in the heart. Empty the blood into tube and reattached syringe to needle. During exsanguination the rabbit will slip deeper and deeper into an anesthetic state resulting in respiratory arrest followed by cardiac arrest after about 90-100cc of blood have been drawn. This will result in death. A total of 120-150cc can usually be obtained (Diano et al., 1987).
The blood will be collected in Microfuge tube. Blood will kept at room temperature for 1 hour so that it can clot. Then centrifuge blood for 5 min at full speed in microcentrifuge. Supernatant will be removed and place in new tube. Centrifuge again for 5 min at full speed. Again supernatant will be removed and place in new tube. Serum will be stored at 5ËšC (or -20ËšC for long term storage). (http://wikilaboratory.com/index.php?title=Serum_Separation)
Bottles containing serum will place in water bath at 56oC and gently swirl the after every 10 minutes until 30 minutes has elapsed. (http://www.thelabrat.com/protocols/heatinactivate.shtml)
Short-term Storage of Serum:
Storage of antibody or of serum (2-3 weeks) at 4Â°C will be carried out.
Long-term Storage of Serum:
For long-term storage, the serum vials will be stored at -20Â°C, which is sufficient for several years. For very long storage serum will be stored at -80Â°C. (http://www.pacificimmunology.com/antibody-storage.asp)
Evaluation of Hyperimmune Serum:
The specific antibodies synthesis in rabbit's serum will be evaluated through agar gel precipitation test as described by Kubey et al., 2008. Results will be analyzed through statistical method.
To calculate the differences between two treatments (0.5 mg/ml and 1 mg/ml of protein), data will be analyzed by Student T- test (Steel and Torrie, 1986).