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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 . 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 .
Gene vaccines are becoming commonplace and have the advantage of raising
antibodies to a target antigen specifically . However, DNA
immunization can stimulate florid local inflammation . DNA vaccines
are commonly delivered in polyethyenimine complexes, where the plasmid
DNA remains active in cells at least 12 days after injection .
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 . 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 . 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
. 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
A recombinant plasmid DNA vaccine was made to control infectious bursal
disease , 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 , 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 . 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
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 . 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 . 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
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 . 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 . 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  (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  (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 , 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
. The sheep nematode parasite, Trichostrongylus, was sensitive to
RNAi . RNAi targeted developmental control genes in chicken embryos
. RNAi could be used to prevent avian influenza . RNAi
specifically silenced genes in fish embryos, and specific gene knockout
appeared effective in medaka, zebra fish and rainbow trout , and was
used to silence the myostatin gene leading to giant zebra fish . The
tiger frog iridovirus also attacks fish; and RNAi was effective in
inhibiting replication of the virus in fish cells .
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 . 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 . 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 . 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 , after a single
injection of the plasmid. Continuous infusion of bovine growth hormone
releasing factor increased milk production by as much as 46 percent
. 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
. 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 . Another vector containing chicken interferon,
when combined with an antigen (sheep red blood cells), resulted in
enhanced antibody response . 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 .
Protozoan predation was the main cause of the introduced bacteria
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 .
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 . The ecology of
the rumen has proved refractory to recombinant bacteria. Genes for
microbial fibrolytic enzymes have been transferred to probiotic bacteria
, 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 .
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