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[SANET-MG] gm food animals part 2

gm food animals part 2
B. Non-heritable modifications of food animals
Non-heritable modifications of food animals include a number of applications such as DNA vaccination, transgenic probiotic bacteria as vector for vaccines and growth hormones, using RNAi (RNA interference) for epigenetic modifications, and stem cell chimeric animals whose somatic tissue but not the germ cells are transgenic. Non-heritable alterations are taking place or being implemented without full review of the impact on food and the environment, mainly because they do not fall under the rubric of genetic modification.

Naked DNA vaccines
It has been shown since the 1990s that ingested foreign DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice [31]. Since then, naked DNA has found many applications, especially as DNA vaccines. DNA vaccines can be applied by a variety of routes including intradermal, intravenous, intramuscular, intraperitoneal, subcutaneous, sublinqual, intravaginal, intrarectal, via internasal inhalation, intranasal instillation, ocular and biolistic delivery [32]. Gene vaccines are becoming commonplace and have the advantage of raising antibodies to a target antigen specifically [33]. However, DNA immunization can stimulate florid local inflammation [34]. DNA vaccines are commonly delivered in polyethyenimine complexes, where the plasmid DNA remains active in cells at least 12 days after injection [35]. DNA vaccines are used in both farm animals and fish, and there does not appear to be reports on whether there is any carry over of the vaccine DNA into food prepared from vaccinated animals. Pigs have been immunized against pork tapeworms with a DNA vaccine injected intramuscularly [36]. Pork tapeworm can be transmitted to humans. An improved DNA vaccine for bovine herpes virus-1 was constructed using the viral gene for protein VP22 [37]. Bovine herpesvirus 1 (BHV-1) causes several diseases in cattle worldwide, including inflammation of nose and trachea, vagina, penis, eyes, gut, and abortion. BHV-1 is also a contributing factor in shipping fever. It is spread through sexual contact, artificial insemination, and aerosol transmission. A novel combination of recombinant DNA and recombinant protein was used to vaccinate cattle against mastitis caused by Staphylococcus aureus [38]. The cattle were first immunized with two recombinant DNA plasmids; the first containing gene fragments of the fibronectin binding motifs joined to gene fragments of clumping factor A, the second carrying a gene for the bovine granulocyte-macrophage - colony stimulating factor gene. Pregnant heifers were immunized twice with the DNA plasmids then boosted with the recombinant hybrid protein consisting of fibronectin binding motifs joined to fragments of clumping factor A. The immunized heifers were partially protected from mastitis and illnesses after infection. A recombinant plasmid DNA vaccine was made to control infectious bursal disease [39], a highly contagious viral disease of young chickens characterized by immunosuppression and mortality generally at 3 to 6 weeks of age. The vaccine plasmid contained the VP2 gene of the double stranded RNA virus driven by the human cytomegalovirus immediate early enhancer and promoter, the adenopartite leader sequence and a SV40 polyadenylation signal. The plasmid vaccine also contained a CpG oligonnucleotide adjuvant to enhance innate immunity. The combined vaccine was reported to effectively control infectious bursal disease. A recombinant plasmid DNA vaccine was prepared to control viral hemorrhagic septicemia [40], a systemic infection of various salmonid and a few nonsalmonid fishes caused by a rhabdovirus (a single stranded RNA virus). The virus infection occurs in fish of any age and may result in significant mortality. The plasmid vaccine contained a recombinant glycoprotein gene from the virus; and specific antibodies against the recombinant protein were detected after vaccination. A DNA vaccine was made to protect against Mycobacterium marinum that causes tuberculosis in fish and shellfish and cutaneous lesions in humans [41]. The bacterium is transmitted from fish to humans. The vaccine consists of a DNA plasmid carrying the bacterial gene for a protein that binds to a secreted fibronectin. Fibronectin is a high molecular weight fish glycoprotein that binds to receptor proteins called integrins spanning the cell membrane. In addition to integrins, they also bind to extracellular matrix components such as collagen, fibrin and heparin. Vaccinated striped bass were protected from the bacterium.

Recombinant vaccine vectors
Recombinant vectors have been developed from viruses or bacteria to deliver vaccine antigens. One fundamental concern over the use of such vectors is genetic recombination involving the vectors, resulting in novel pathogens. Not only are the vectors themselves already derived from pathogens, but they also carry transgenes from other pathogens. A Newcastle disease virus was modified to express the H5 hemagglutinin of avian influenza. Newcastle disease is a highly contagious bird disease affecting many domestic and wild avian species, and is caused by a single stranded RNA virus. Its effects are most notable in domestic poultry, which are highly susceptible to the disease with the potential for severe epidemics that impact on the poultry industry. Avian influenza is endemic to many countries, and is a threat to both commercial and wild fowl as well as to humans. The virus can change to a form that causes serious disease in humans through reassortment, mutation and recombination [42-44] (Fowl Play in Bird Flu; Where's the Bird Flu Pandemic?; What Can You Believe About Bird Flu?). The chimeric vector vaccine is expected to protect against both influenza and Newcastle disease. The vaccine was tested so far only on about 15 chickens that were examined after 10 days and judged healthy [45]. It is clear that more extensive safety studies are needed. A recombinant pseudorabies virus expressing a fusion protein of pig circovirus type 2 was made [46]. Pseudorabies viral disease in swine is endemic in most parts of the world, and is caused by porcine herpesvirus 1. The name pseudorabies comes from the similarity of symptoms to rabies in dogs. Secondary hosts are infected through direct contact with swine, or via infected pork. Porcine circovirus (PCV) is a member of the virus family Circoviridae; and there are two serotypes, PCV1 and PCV2. These relatively small, non-enveloped, circular DNA viruses are quite stable in the environment and resistant to many common disinfectants. PCV2 is associated with postweaning multisystemic wasting syndrome (PMWS) in piglets, characterized by progressive loss of body condition, visibly enlarged lymph nodes, difficulty in breathing, and sometimes diarrhoea, pale skin, and jaundice. The vaccine appears to protect against both circovirus and psuedorabies virus infection, but its safety remains to be ascertained. The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals appears promising. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. By the same token, genetically modifying these bacteria has the potential to turn them into serious pathogens. Lactobacillus isolated from the gastrointestinal tract of broiler chickens and selected for probiotic characteristics was genetically modified by inserting an expression cassette into the lbs gene [47]. The transformed bacteria expressed different fluorescent cell surface proteins used as reporters of promoter function. It is possible that the same procedure can be used to construct bacteria expressing pathogen antigens as live oral vaccines to immunize broilers against infectious diseases. A number of such oral vaccines have been successfully tested in mice but reports of vaccination of food animals are not yet available. Using GM probiotic bacteria as vaccine vectors requires special caution. These bacteria are natural beneficial symbionts of the gastrointestinal tract, and have adapted to their human and animal hosts over millions if not billions of years of evolution. Genetically modifying them as vectors could easily turn them into pathogens pre-adapted to invade the human and animal gut [48]. Furthermore, the gastroinstestinal tract is an ideal environment for horizontal gene transfer and recombination, the major route to creating pathogens. For these reasons, we have proposed that any genetic modification of probiotic bacteria should be banned [49, 50] (Ban GM Probiotics; GM Probiotic Bacteria in Gene Therapy). There is increasing evidence that infectious disease epidemics, such as bird flu, are created by intensive industrial farming of livestock and the globalised trade in livestock, meat and animal products [42] (Fowl Play in Bird Flu). Vaccines are risky on the whole, and cost a lot to develop; and may well not be necessary if much more effort were devoted to establishing farming practices that reduce stocking rates while improving animal welfare, nutrition and health to build up the animals’ natural immunity to disease.

RNAi in epigenetic gene modification in food animals
Among the major discoveries of molecular genetics in the 1990s is RNA interference (RNAi), how very small RNA molecules - around 21 to 25 nucleotides or shorter - can inhibit expression of specific genes in all organisms [51] (Subverting the Genetic Text). RNAi regulates basic biological processes, including transition from one stage of development to another. Furthermore, RNAi is used as a form of immunity to protect the cell from invasion by foreign nucleic acids introduced by mobile genetic elements and viruses. RNAi soon found applications in human gene therapy [52], as it appeared to offer the ability to shut down any chosen gene specifically without affecting any other. But the technique hailed as “breakthrough of the year” in 2002 was found not to be so specific after all. There were substantial “off target” effects on other genes and proteins [53, 54] (Controversy over Gene Therapy 'Breakthrough'). In May 2006, RNAi gene therapy was found to kill mice by the dozens [55, 56] (Gene Therapy Nightmare for Mice). The mice died of liver failure from RNAi overload. There are reasons to believe that RNAi therapy is unsafe, because the effects are not, and cannot be specific. Numerous RNA species interfere at every level of gene function, and it is impossible to target the effects precisely because the RNA interference underworld is huge, comprising some 97 to 98 percent of the transcription activity in the cell, and specificity depends on low levels of the correct sequences being produced at the right time in the appropriate places. Extreme caution is needed as these RNAi species have the potential to affect the animals adversely, and can also be passed onto humans through food. RNAi has been used as a tool to study gene function in bovine oocytes. The percentage of active oocytes was increased following RNAi treatment [57]. The sheep nematode parasite, Trichostrongylus, was sensitive to RNAi [58]. RNAi targeted developmental control genes in chicken embryos [59]. RNAi could be used to prevent avian influenza [60]. RNAi specifically silenced genes in fish embryos, and specific gene knockout appeared effective in medaka, zebra fish and rainbow trout [61], and was used to silence the myostatin gene leading to giant zebra fish [62]. The tiger frog iridovirus also attacks fish; and RNAi was effective in inhibiting replication of the virus in fish cells [70].

Somatic gene therapy in farm animals using vectors or naked DNA
Gene therapy has been used in farm animals to transform somatic cells without affecting the germ cells, at least in theory. Retrovirus mediated gene transfer in lungs of living feta sheep has been demonstrated. A Moloney murine leukemia retrovirus vector incorporated a marker gene and either beta-galactosidase, or human interleukin receptor antagonist gene. Gene integration was observed in cells of the airway epithelia [63]. A plasmid vector highly efficient at releasing growth hormone was introduced into the skeletal muscle of pigs using electroporation. The somatic transgenic pigs showed enhanced weight gain and improved body composition at low DNA plasmid dose [64]. An adenovirus vector was used to deliver a human gene angeopoein-1 into the pig heart in animals affected by chronic myocardial ischemia. The implanted gene helped the pigs recover from the condition [65]. A DNA plasmid encoding somatostatin fused with an antigenic protein of a pig reproductive and respiratory syndrome virus induced antibodies to the viral protein and promoted growth in immunized pigs [66], after a single injection of the plasmid. Continuous infusion of bovine growth hormone releasing factor increased milk production by as much as 46 percent [67]. A vector created from the bovine leukemia virus carried the gene for growth hormone release factor driven by a mouse whey acidic protein promoter, or alternatively, a mouse mammary tumour virus promoter; and bovine kidney cells were transfected with the vector. A fowl adenovirus vector was used to insert chicken interferon gene controlled by the fowl adenovirus late promoter and SV40 polyA site [68]. Chickens treated with the recombinant vector showed increased weight gain, and less weight loss when challenged with the parasite causing coccidiosis. A live fowlpox virus vector was constructed carrying a chicken mylomonocytic growth factor gene. Chickens treated with the vector had elevated monocyte levels and a high proportion of active monocytes [69]. Another vector containing chicken interferon, when combined with an antigen (sheep red blood cells), resulted in enhanced antibody response [70]. Using the interferon vector alone increased weight gain and improved resistance to disease.

Recombinant microbes in the rumen
Genetic modification of the microbes in the rumen is a seductive topic. In theory the microbes can be modified to make fodder much more digestible, thus making more efficient use of grazing land. Even though the approach is fairly easy to implement it has not proven effective as yet, because rumen ecology is complex. All too often, the recombinant microbes proved easy prey for the native protozoa of the rumen. On the other hand, if the recombinant microbes succeed, they may unbalance the ecology of the rumen and cause disease to the animals and to the human beings that use the animal and animal products as food. Genetic engineers should learn much more about the ecology of the rumen. A recombinant rumen bacterium, Butyrivibrio fibrisolvens, expressing a fungal xylanase gene and erythromycin resistance marker gene was inoculated into a sheep’s rumen. The recombinant bacterium disappeared from the rumen of hay-fed sheep within 12 hours of being introduced, but flourished when inoculated into autoclaved rumen fluid; showing that the recombinant bacteria were eliminated by living organisms [71, 72]. The main fibre-digesting bacteria in the rumen, Ruminococcus and Fibrobacter, have proved refractory to genetically modification, leaving only Butyrivibrio that can be modified. The recombinant bacteria were less effective at digesting fibre than the native fibre digesters [73]. Protozoan predation was the main cause of the introduced bacteria disappearing [74]. The toxin flouroacetate accumulates to high levels in some Australian plants, becoming lethal to grazing sheep. A gene for flouracetate dehalogenase was isolated from the bacterium Moraxella and used to modify Butyrivibrio fibrisolvens. Sheep exposed to flouracetate showed markedly reduced poisoning symptoms after being inoculated with the recombinant bacteria [75]. In spite of a great deal of effort, recombinant bacteria have not adapted to the rumen. The protozoan residents of the rumen have prevented ready colonization by recombinant bacteria. Interestingly, over 75 percent of the genes for carbohydrate in rumen ciliates originated by horizontal gene from rumen bacteria [76]. The ecology of the rumen has proved refractory to recombinant bacteria. Genes for microbial fibrolytic enzymes have been transferred to probiotic bacteria [77], however. Such efforts could potentially redesign the food animals’ digestive systems. Many of the permanent bacterial residents of the rumen have not yet been cultured. Wild animals may have acquired microbes not seen in domestic animals because they are exposed to more severe dietary conditions. Such microbes and their enzymes may be useful for applications in the future [78].

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