Biology Project

Biologically Modified
Organisms
Omar Jameel
XII-B Boys
 Acknowledgment

I would like to express my deepest gratitude

towards my biology teacher Mrs. Seema Gupta
and the principle Mr. Rishikesh Padagonkar for
presenting me with this wonderful opportunity
of working upon this project of the topic,
Biologically Modified Organisms.
It has really allowed me to expand my horizons,
learn new things and delve even further into
the world of biology.
And lastly, I would like to thank my friends and
family for supporting and encouraging me
throughout the course of this project.
 Index
Topic Pages
Introduction 4-7

How did it Begin? 8-10

Current Working 11-12

Safety Assessment of GM Food or
Feed 13-15
Immunotoxicity and Food Allergen
16-17
Immunogenecity and Adjuvanicity
18-22
Allergenecity and Immunotoxicity
in Animals 23-40
Horizontal Gene Transfer 41-56
Lower Mytoxin Levels and GM 57-63
Crops
Gm Stacked Events 63-76
Nutritionally Altered GM Crops
77-110
Case Studies 111-124
Bibliography 125
 Introduction
Biologically modified organisms have a very large scope and its
definition can greatly vary but it generally refers to those organisms
which have had their genes altered naturally or artificially.
A more specific definition is being used by the World Health
Organization (WHO) and the Food and Agriculture Organization (FAO)
where they say that the term ‘Biologically Modified Organisms’ is only
applied when the reproduction of living organisms takes place without
any form of natural reproduction.
Genetically modified organisms are also known as genetically
engineered organisms when they are made by the use of gene
manipulation techniques, where the genes of an organism are altered,
new genes from another organism are introduced or even when
unnecessary genes in an organism are removed.
Creating a genetically modified organism is a multi-step process.
Genetic engineers must isolate the gene they wish to insert into the host
organism and combine it with other genetic elements, including a
promoter and a terminator region and often a selectable marker. A
number of techniques are available for inserting the isolated gene into
the host genome.
Bacteria are the easiest class to test and experiment these techniques
upon, and thus were the first organisms to be genetically modified. After
simple testing they were modified with the hope of using them mainly
for environmental or medicinal purposes.
They were also used for industrial protein purification. Fungi have also
been modified with similar goals.
Viruses play an extremely important role as vectors in the process of
adding genetic material into other microorganisms. There have also been
proposals of removing the virulent segments of a virus's genetic code so
as to make vaccines.
Plants have also been genetically modified for better crop yield, more
nutritional value, more forgiving growth conditions, better disease and
pest resistance and shorter growth time. There are also promising
researches where genetically modified plants are being made as
bioreactors for the production of biofuels, medicines and
biopharmaceuticals.
Genetically modified crops have been a very controversial topic ever
since they came into existence. Many people believed that these new
crops were actually not fit for consumption and there were also people
who rejected them on the basis that they were something that went
against their religion, that is, blaspheming the creation of God in order to
satisfy our own primal needs.
Animals are much harder to genetically modify and thus, the process is
still in its research stage. Keeping the possibility of human genetical
modifications in mind, mammals are the best species to experiment upon
right now due the genetical and physical similarity they have with
regards to us human beings.
If the genetical modification of humans, one day became safe and easy
to do it would open up an astounding number of possibilities to us. We
would be able the genetically removes diseases from patients, have
better disease resistance, gain characteristics that are useful for us in our
environment and even gain longer lifespans.

Right now, genetical modifications are used upon animals to give better
growth rates, better meat quality, good milk composition, disease
resistance, natural products (clothing material, eggs, etc.) and
survivability in general.
Biological modifications have also been proposed to control the spread
of mosquitos, a vector for many diseases.
Although human gene therapy is still new and in its experimental stages
it has been used to cure genetical disorders such as severe
immunodeficiency disease, and Leber’s congenital amaurosis.
Before the commercialization of genetically modified organisms was
made possible on a large scale there were numerous tests that needed to
be done to ensure that they were safe for consumption and also look
upon the concerns that people had regarding the fact genetically
modifying some organisms for our benefit would negatively affect them
by changing their life style in such a way that they could not cope with it
outside of supervised growth. We also needed to check upon the changes
these modifications would bring to other organisms and nature in
general and see if there was any way to minimalize these changes.
Thus, there are many differences in the regulation of genetically
modified organisms, mainly Europe and the US. One of the key
concerns is whether genetically modified foodstuff should be labelled
and categorized as gene edited organisms.
 How did it Begin?
Humans have domesticated plants and animals since around 12,000
BCE, using selective breeding or artificial selection. The process of
selective breeding in which organisms with desirable traits ( desirable
genes) are used to breed the next generation and organisms lacking the
specific trait are not bred , is nothing but a precursor to the concept of
modern day genetic engineering Various advancements in the field of
genetics now allow us to modify and directly alter the DNA of
organisms into making them possess the traits that we want them to
have.

Most noticeably: CRISPR, a simple but powerful gene editing tool can
use the protein “Cas9” as a pair of molecular scissors to freely cut
segments of DNA, have made the production of genetically modified
organisms much simpler than before. The first genetically modified
organism, a bacterium resistant to the antibiotic kanamycin was made by
Herbert Boyer and Stanley Cohen in 1973. The first genetically modified
animal was a mouse made by Rudolf Jaenisch in 1974 by inserting
foreign DNA into it as an embryo. The mice which were genetically
engineered were used to produce a human tissue plasminogen activator,
a protein involved in the breaking up of blood clots and the first
genetically modified plant was made in 1983 by Michael W. Bevan,
Richard B. Flavell and Mary-Dell Chilton They infected tobacco with
Agrobacterium transformed with an antibiotic resistance gene and
through tissue culture techniques were able to grow a new plants
containing the same resistance gene.
The first plant to be engineered purely for the enhancement of nutritional
value was Vitamin A enriched golden rice. The first genetically
modified antibiotic resistant plant was tobacco in 1982. China was the
first country to commercialize a genetically modified plant in the form
of a virus resistant tobacco plant in 1992.
In 1994 Calgene obtained the permission required for the commercial
release of Flavr Savr tomato, the genetically modified food. Also, in
1992 the European union approved of the production of tobacco
engineered to be resistant to the herbicide bromoxynil, making it the first
commercialized crop in Europe. An insect resistant potato plant was
given approval in the US in 1995 and by 1996 approval had been
granted to 8 genetically modified crops and one genetically modified
flower in six countries plus the European Union.

Golden rice (left) and normal rice (right).

In 2010, scientists at the J. Craig Venture Institiute announced that they
had created the world’s first ever synthetic bacterial genome called
Synthia. It was the world first ever synthesized lifeform. The first
genetically modified animal to be commercialized was the GloFish, a
zebra fish with a fluorescent gene added that allowed it to glow in the
dark under the exposure of ultraviolet light. It was released to the market
in 2003. In 2015 AquaAdvantage salmon become the world’s first ever
genetically modified animal to be approved for consumption. This
approval is for the fish raised in Panama and sold in the United States.
The salmon were transformed with a growth hormone regulating gene
form a Pacific Chinook salmon and a promoter from an Ocean Pout
which allowed it to grow all year round instead of inly during spring and
summer.

Synthia, Glofish and AquaAdvantage Salmon.

 Current Working
CRISPR-Cas9 is short for clustered regularly interspaced short
palindromic repeats and CRISPR-associated protein 9. The CRISPR-
Cas9 system has generated a lot of excitement in the scientific
community because it is faster, cheaper, more accurate, and more
efficient than other existing genome editing methods.
CRISPR-Cas9 was adapted from a naturally occurring genome editing
system in bacteria. The bacteria capture snippets of DNA from invading
viruses and use them to create DNA segments known as CRISPR arrays.
The CRISPR arrays allow the bacteria to "remember" the viruses (or
closely related ones). If the viruses attack again, the bacteria produce
RNA segments from the CRISPR arrays to target the viruses' DNA. The
bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which
disables the virus.
The CRISPR-Cas9 system works similarly in the lab. Researchers create
a small piece of RNA with a short "guide" sequence that attaches (binds)
to a specific target sequence of DNA in a genome. The RNA also binds
to the Cas9 enzyme. As in bacteria, the modified RNA is used to
recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the
targeted location. Although Cas9 is the enzyme that is used most often,
other enzymes (for example Cpf1) can also be used. Once the DNA is
cut, researchers use the cell's own DNA repair machinery to add or
delete pieces of genetic material, or to make changes to the DNA by
replacing an existing segment with a customized DNA sequence.
Genome editing is of great interest in the prevention and treatment of
human diseases. Currently, most research on genome editing is done to
understand diseases using cells and animal models. Scientists are still
working to determine whether this approach is safe and effective for use
in people. It is being explored in research on a wide variety of diseases,
including single-gene disorders such as cystic fibrosis, hemophilia and
sickle cell disease.
It also holds promise for the treatment of more complex diseases, such
as cancer, heart disease, mental illness and human immunodeficiency
virus (HIV) infection. Ethical concerns arise when genome editing,
using technologies such as CRISPR-Cas9, is used to alter human
genomes. Most of the changes introduced with genome editing are
limited to somatic cells, which are cells other than egg and sperm cells.
These changes affect only certain tissues and are not passed from one
generation to the next. However, changes made to genes in egg or sperm
cells (germline cells) or in the genes of an embryo could be passed to
future generations. Germline cell and embryo genome editing bring up a
number of ethical challenges, including whether it would be permissible
to use this technology to enhance normal human traits (such as height or
intelligence). Based on concerns about ethics and safety, germline cell
and embryo genome editing are currently illegal in many countries.
 Safety Assessment of GM Food or Feed
Food/feed allergy is a pathological deviation of the immune response.
Most frequent and severe allergic reactions in humans are mediated by a
particular class of immunoglobulins called IgE antibodies. In allergic
individuals, minute amounts of a food that is well tolerated by most of
the population can cause severe symptoms and even death. However,
food allergy is different from toxicity and intolerance. Toxicity is
reproducible, predictable and affects all individuals exposed with only
minor inter-individual differences in susceptibility.
There is a clear direct dose-response relationship between the dose of
exposition to a toxic and the severity of the reaction. Food intolerance
may affect large populations of individuals who are genetically deficient
for one particular enzyme such as lactase, which is missing, particularly
in most population of human adults.
This results in an impaired digestion of lactose and leads to the
production of lactic acid by colonic bacteria, which causes abdominal
pain, bloating and diarrhea. In many cases, the mechanisms for food
intolerance are not all well understood.
Food poisoning may occur when high concentrations of bioactive
substances such as histamine produced because of microbial
contamination, are present in adulterated food (e.g. fishes).

Classification of adverse reactions to foods.

Also, symptoms of food intolerance may be similar to those observed
during an allergic reaction; they are due to the same pharmacologically
active mediators although no immune mechanisms are involved for their
synthesis and release from the same cells (e.g. mast cells or basophils);
such reactions are called pseudo allergy.
Food/feed allergy, in contrast, is a pathological deviation of the immune
response to a particular substance, which affects only some individuals
where a combined effect of variations in the environment and genetic
predisposition has resulted in allergic sensitization. In allergic
individuals, minute amounts of a food that is well tolerated by the vast
majority of the population can cause severe symptoms and even death.
During the risk assessment, and allergenicity of GM plants and derived
food and feed is assessed on the basis of structural characteristics and
the physical and biochemical properties of the newly expressed protein
using in silico, in vitro and in vivo tests according to appropriate
guidelines.
 Immunotoxicity and Food Allergy
Food allergy is an immune adverse reaction to some food proteins or
food protein fragments that are well tolerated by the general population.
Allergic reactions to these food proteins are mediated by type 1
immediate hypersensitivity reactions (IgE-mediated), delayed
hypersensitivity (reaction associated with specific T lymphocytes) and
inflammatory reactions caused by immune complexes. The most
common reaction to food allergens is immediate hypersensitivity, which
can be divided into two phases: sensitization and the allergic response.
Sensitization occurs when the antigens pass the mucosal barrier. They
are taken up and carried on the surface of antigen presenting cells
(APC), the APCs present the antigen to T cells.
The binding to T cell receptors occurs if the processed antigen is
presented in combination with specific molecules of the major
histocompatibility complex (MHC). Different T cell subclasses are then
activated which results in different kinds of immune responses. In
particular the production of specific antibodies of the IgE class may be
induced. This is what happens during the sensitization phase. The IgE
produced circulate and bind to the membrane of immune cells such as
blood basophils and tissue mast cells. No clinical manifestation occurs
during the sensitization. Upon a subsequent exposure to the same or to a
structurally similar protein, more specific IgE antibodies are produced.
They also bind on the basophils and mast cells. The responsible protein,
then called allergen, circulates in blood and bind to the IgE antibodies
fixed on the cells in a way that activates a sequence of intracellular
events which result in degranulation of cells and release of preformed
and newly formed pharmacologically active mediators such as
histamine, prostaglandins and leukotrienes which causes symptoms
(allergic reaction). Clinical manifestations are various: they can affect
the gut (e.g. diarrhea) but also the skin (atopic dermatitis) and
respiratory system (asthma) and even provoke a severe and possibly
fatal systemic anaphylactic reaction.The food proteins or food protein
fragments that induce an allergic reaction commonly escape extensive
hydrolysis during gastric and intestinal digestion.
In humans, food allergy is mostly observed in the first months and years
of life and decreases with age. Digestive exposure to allergenic protein
mainly occurs during the neonatal period before complete closure of the
intestinal barrier and while the immune system and particularly the gut-
associated lymphoid tissue (GALT) is still immature. It is noteworthy
that sensitization may also occurs by contact with the skin and by
inhalation.
The permeability of the intestinal mucosa to proteins can be increased by
infectious agents or toxins but can also result from local hypersensitivity
reactions in the gut. Allergic responses may also occur in large domestic
farm animals, especially calves and piglets at weaning but not in
ruminants where most potentially allergenic proteins are degraded in the
rumen.
 Immunogenicity and Adjuvanticity
Immunogenicity and adjuvanticity are properties of food/food
constituents that may be indirectly related with the sensitizing potential
and are therefore assessed, where deemed necessary, in the same context
as allergenicity.
Immunogenicity is the capacity of a protein to induce an immune
response after exposure under appropriate conditions in humans and
animals. In general, this will result in the production of the different
classes of antibodies among which IgE are present in very small
concentration. Peptide fragments generated after proteolytic cleavage of
the protein may retain (part of) the immunogenicity. Allergenicity is a
particular pathological form of immunogenicity which associates the
structure of the involved protein, the genetic background of the
individual and particular environmental conditions for the exposure.
Adjuvants are substances (e.g. food component or possibly newly
expressed proteins in GMOs) that, when co-administered with an
antigen (e.g. protein), increase the immune response to that antigen. It
can thus be expected to increase also the development of allergy against
bystander proteins present in the environment. This can be achieved by
many different pathways. The adjuvant is generally not immunogenic by
itself but it may alter the immunological status of the exposed
human/animal. They may also activate NK and NKT cells resulting in
increased pathogenicity in absence of any antigen.
In addition to substances having a direct adjuvant effect, other
substances may indirectly have a similar effect by reducing food protein
enzymatic degradation or by increasing intestinal mucosa permeability
and thus antigen uptake (e.g. saponinsin foods).
In animal models, experimental sensitization using adjuvant increases
the specific IgE response and may lead to an allergic reaction that causes
adverse effects of different degrees of severity. Adjuvants-may also
induce an increased production of antibodies of other isotypes than IgE;
as an example, an increased specific IgA antibody response could be
expected to be protective at the mucosal level both in airways and
digestive exposures.
Denaturation during heat treatments and/or degradation by digestion
may alter the immunoreactivity and adjuvanticity of a newly expressed
protein as a consequence of alteration of its structure and biological
activity as compared to the native protein; however, this may not be a
general rule and for instance, receptors for innate immunity detect also
denatured adjuvants.
In the case of immunotoxicity, differently to the antigen-specific
immunological process of allergy, food substances can exert toxic
effects that specifically affect the immune system. They generally result
in a decreased protective response and a higher susceptibility to diseases.
Feed allergens:
Food-associated antigens that cause allergy have mainly been described
in man. Most common allergens for humans are found in cow's milk, soy
bean, chicken meat, beef, eggs and gluten from wheat, oats, rye and
barley. For some foods, particularly milk but also egg and even peanut
resolution of food allergy often occur spontaneously by the age of 3. The
maturation of the gut immune system, an increase in the number of
regulatory T cells and modification of the composition of the gut
microbiota due to qualitative changes in the diet are likely to be involved
but the mechanisms are not fully understood.
 Some common food allergens.
From the compositional analyses of GM crop varieties known to be
allergenic that have been assessed for market approval for the European
Union in the last few years (i.e. GM soybean) there so far have been no
indications that the crops may comprise increased levels of allergens.
In calves and pigs, only allergies to soybean proteins have been reported
under natural conditions.
Although naturally occurring feed allergies in livestock animals are
reported, epidemiological data are scarce and information on the inter-
herd or intra-herd prevalence is not available. Especially pigs have been
used to study the pathogenesis and immune responses to allergens. Pigs
present important advantages compared with other animal models; they
closely resemble humans in gastrointestinal physiology and in the
development of mucosal immunity. Developing piglets have similar
anatomy and nutritional requirements to that of the developing infant, a
transient neonatal porosity to the gut to dietary proteins, a distribution
and maturation of intestinal enzymes, and an enteric absorption of
antibody.
They are born immunocompetent, allowing for assessment of immune
responses. Furthermore, pigs, and also calves, demonstrate similar
induction of hypersensitivity responses to those of human allergic
disease. Pigs have an IgE-mediated-like response to parasites, legumes,
and pollens comparable to that in. More information on food allergy in
livestock is available after sensitization and challenge experiments.

One of the major components of animal feed in the EU, soy bean, has
been described to be allergenic for calves and pigs. Soybean proteins can
induce poor growth performance, diarrhea, serum and intestinal IgG
anti-soy antibodies, higher concentration of IgE in intestine and serum,
reduced intestinal flow rate, atrophy of intestinal villi or lower villi
height, skin test positivity and increased small intestinal mucosal and
submucosal mast cell numbers in previously (mostly about 2 weeks of
age) sensitized pigs and calves.
Clinical features of feed allergy:
In humans, food allergy comprises a wide spectrum of symptoms located
in the gastrointestinal tract (gastrointestinal food allergy) and/or
elsewhere in the body, such as the respiratory system, skin or eyes (food
allergy).
This may be combined in one patient, resulting in a vast range of clinical
manifestations, from acute anaphylactic, generalized reactions, even
leading to death, to relatively minor local signs like diarrhea or
abdominal pain.
 Allergenicity and Immunotoxicity in
Livestock Animals
Calves:
Immune-mediated gastro-intestinal disorders were reported in calves fed
milk formulas containing heated soybean flour. The major clinical signs
were loss of appetite, diarrhea and body weight reduction. They were
likely resulting from either type I (IgE-mediated) or type III (immune-
complex mediated) hypersensitivity reactions.

The very rapid (<1 h) onset of motility disturbances of the small

intestine following a challenge with antigenic soybean in calves already
sensitized to this food was also consistent with an IgE-mediated
reaction. Histamine appeared to be the main mediator involved in
motility disorders. Sensitive calves developed very high levels of serum
IgG1 antibodies mainly against major soybean proteins, namely glycinin
(11S globulin) and β-conglycinin (7S globulin) but other minor proteins,
including α-conglycinin, Bowman-Birk inhibitor and lectin also induced
skin reactions. β-Conglycinin, but not glycinin was also able to induce in
vitro proliferation of lymphocytes collected from sensitized calves.
This, together with long-lasting skin reactions after intradermal injection
of β-conglycinin, suggested that this protein is involved in type IV,
cellular-based hypersensitivity, reactions in calves (Dreau and Lalles,
1999). Histology revealed strong intestinal villous atrophy associated
with increased permeability. Increased mast cell numbers and eosinophil
infiltration of mucosal tissues with T lymphocyte subsets was also
reported in sensitive calves (Dreau and Lalles, 1999). β-Conglycinin was
shown to resist gastric digestion in vivoand both glycinin and β-
conglycinin were detected in digesta in the small intestine. Appropriate
processing of soybean products (e.g. using heat treatment, proteases or
hot water-ethanol mixture) reduced considerably clinical adverse
manifestations to feed and improved growth performance. It has been
proposed that the immunogenicity of soybean products, their
digestibility and nutritional value for growing calves could be predicted
from the levels of immunoreactive glycinin and β-conglycinin in
soybean products.
Beside soybean, veal calves were shown to digest raw field pea flour
poorly and to develop severe immune-mediated gastro-intestinal
disorders and diarrhea under normal rearing.

Pigs:
Allergy to feed (e.g. soybean), suspected to be involved in the post-
weaning diarrhoea syndrome in young pigs has been well documented
(Dreau and Lalles, 1999 for review). Serum antibodies to food were
consistently observed in pigs fed antigenic soybean products after
weaning. Specific IgM and IgG antibodies were found in plasma, and
IgM, IgA and IgG in intestinal secretions, in piglets fed proteins that
were still antigenic (e.g. in a non-denatured form).
Histological investigations demonstrated an infiltration of the intestinal
mucosa by T cells following antigenic soybean consumption. As
described for calves, the respective roles of major storage proteins, e.g.
glycinin or β-conglycinin, have been investigated and an IgE-mediated
reaction to glycinin was demonstrated. Gastric administration of glycinin
was able to induce an IgE-mediated response in pigs in a dose-dependent
manner, resulting in diarrhea and reduced growth performance. This
allergic response was associated with increased numbers of intestinal
mast cells, increased histamine release and increased plasma
concentrations of IL-4 and IL-10. It has been shown that both soybean
globulins were digested along the small intestine. However, β-
conglycinin was found in higher concentrations than glycinin in the
caecum and colon, indicating a higher resistance to digestion. Purified
glycinin incorporated into the diet resulted in diarrhea and reduced
growth performance. It also increased lympho-proliferation, IgA
antibody concentrations, IL-4 and IL-6 concentrations and mast cell
counts in jejunal mucosa of sensitized pigs. Finally, plasma anti-glycinin
IgE antibodies and intestinal mast cell numbers increased linearly with
increasing glycinin concentrations in pig diet.
Mini pigs sensitive to glycinin displayed lower jejunal and ileal mast cell
densities than those sensitive to β-conglycinin (and non-sensitized
controls). The gut microbiota may play a role since piglets reared in an
isolator displayed higher anti-soybean antibody responses after weaning,
showing the importance of early-life environment in the onset of food
allergies in pigs.
Another important finding is the identification of a major allergen from
maize for young pigs. It is a storage protein from the prolamin family
classified as γ-zein with a molecular mass of 27 kDa and shown to be
resistant to pepsin digestion. This protein cross reacts with other proteins
having in common large conserved poly-glutamine domains and also has
sequence similarity with many other food allergens (e.g. glutenin, γ-
gliadin, 2S albumin, from different seeds).
Finally, other proteins from legume grains are immunogenic in pigs but
adverse food-related reactions have not been reported against these
legume proteins.

Fish:
Adverse reactions to soy proteins have been suspected to cause
deleterious effects on the distal intestine of rainbow trout and Atlantic
salmon. However, little evidence is available to conclude on immune-
mediated, especially IgE-dependent mechanisms. Impacts of high
soybean contents in the diets were observed on the non-specific
immunity response in rainbow trout and on the appearance of alterations
in distal intestinal tissues in salmon.
This may possibly indicate an inflammatory or hypersensitivity response
but no circulating specific antibody against dietary soybean proteins
were found. However increased levels of IgM and T-cell-like responses
to soybean were observed in intestinal tissues of salmon. A possible
cause may be the presence of lectin that binds intestinal epithelium and
cause its disruption in rainbow trout.
Variation in susceptibility to feed allergens:
The balance between tolerance and sensitization depends on several
factors, such as genetic background, nature and dose of antigen,
frequency of administration, age at first antigen exposure, microflora of
the gut, immunologic status of the host, and antigen transmission via
breast milk.
Young animals are in general more susceptible to food allergy than older
animals (Bailey and Haverson, 2006). Feeding unprocessed soy to calves
is associated with persistent expression of active IgG antibody responses
to soy proteins. Piglets weaned onto soy-or egg-based diets developed
antibodies levels comparable to those after primary systemic injection
(Bailey and Haverson, 2006). The young piglets can develop a transient
hypersensitivity response to soybean proteins resulting in diarrhea and
reduced growth rates. Tolerance takes some time to develop, both in
piglets abruptly or gradually weaned on a soy-based diet (Bailey and
Haverson, 2006). Colostrum and maternal milk have effects on the
development of tolerance. Both antibody and antigen (and even immune
complex) can be transferred via milk and affect immune responses of the
neonate. Piglets of sows fed an ovalbumin-containing diet during
gestation and lactation had reduced levels of diarrhea when weaned on
egg-containing diets. This may be the result of the transfer of antigen
itself, because new-born piglets given soy protein by stomach tube also
had a reduced antibody response when subsequently weaned on soy
diets (Bailey and Haverson, 2006).
The dose of antigen played a role in the development of the allergy.
Higher doses have been reported to cause more severe symptoms.
However, other studies describe that pre-weaning exposure to small
amounts of dietary protein sensitize, while feeding (continually) higher
doses of soy-based food prior to weaning prevented adverse effects of
soy-rich post-weaning rations and caused tolerance.
In a physiological situation trans-epithelial antigen transfer in the
intestinal tract is mainly organized via specialized epithelial cells, the M-
cells, but change in epithelial integrity and dysfunctions of the intestinal
epithelial barrier can result in increased antigen translocation. Currently,
no information is available on the effects of high (growth) production
levels or subclinical intestinal infectious diseases in species like chicken,
pigs and veal calves, which might impair intestinal integrity and result in
macromolecular transport and where sensitizing and development of
hypersensitivity cannot be excluded.
As a conclusion, in most animals, including humans, the development of
food allergy is much affected by environmental conditions and
particularly by windows of exposure to the antigens (e.g. age,
physiological status, routes and doses of first exposures).

Diagnosis of food/feed allergy:

Several immunologic methods have been used to make a diagnosis of
food allergy. In the skin prick test, a few drops of the purified allergen
are intradermal injected in the skin, and the skin is monitored for rash or
thickening. Skin prick tests have been shown to have a high negative
predictive value for major foods compared with a low positive predictive
value. This is often variable with age of the individual and quality of the
food extracts. Similar to skin testing, in vitro testing for allergen-specific
IgE (RAST, EAST) is quite variable, especially with the quality of food
extracts and the interference from high total IgE and IgG.

It is well-known that IgE-binding to food proteins may occur in persons

that are clinically tolerant to the food from which they are derived
(Poulsen, 2004).
In humans, the double-blind placebo-controlled food challenges
(DBPCFC) is considered the gold standard for diagnosing adverse food
reactions caused by any mechanism but it may cause severe reactions in
highly allergic individuals. In humans, the combination of a good
clinical history, skin tests, allergen-specific IgE levels, and where
possible the DBPCFC, could be used for the diagnosis of a food
component as the agent causing an adverse IgE-mediated event.
In pre-ruminant calves work has been done to detect serum IgE
antibodies against soy components using the passive cutaneous
anaphylaxis (PCA) test or enzyme immunoassays.
Diagnosis of allergic reactions of livestock to feed is difficult and based
on clinical history and clinical symptoms of the animal. The above
described assays could be used but require availability of (purified)
allergenic proteins. Furthermore, today's livestock feed is so complex
that determination of the exact antigen is nearly impossible.
Allergenicity and Immunotoxicity of GM Products in Livestock
Animals:
In experimental studies allergenicity and immunotoxicity of GM
products has been addressed in laboratory animals (i.e. rodents). The Bt
protein Cry1Ac, which is expressed in some insect resistant GM maize
and has a structure very close to that of Cry1Ab which is expressed in
MON 810, has been shown to be immunogenic in BALB/c mice,
inducing a systemic and a mucosal immune response with production of
specific IgG, IgM and IgA antibodies after intra peritoneal, intra gastric,
intra-nasal or intra-rectal administration. The N-terminal region of
Cry1Ab, Cry1Ac and also of Cry1Aa, another similar protein, may
induce strong specific antibody responses after intra-nasal or i.p.
administration to BALB/c mice, and Cry1Aa was shown to induce
cytokine production in mouse lymphocytes.
The induction of a specific immune response was also observed in rats
after ingestion of transgenic rice containing or spiked with Cry1Ab
protein and 2 human T-cell epitopes of Cry1Ab have been identified in
vitro. In addition, a study suggested that feeding weaning and old mice
with MON810 maize may result in alterations of intestinal and
peripheral immune cell populations.
All these studies demonstrate the intrinsic immunogenicity of Cry1A
proteins but specific IgE were not observed in animals experimentally
treated and also never detected in sera from humans with various
allergies who might have been exposed to insect-resistant crops.
Changes in the structure (e.g. post-translational modifications) of the
expressed trait protein may result in unintended effects on the immune
response; it has thus been reported that the recombinant form of the bean
α-amylase inhibitor was expressed in a GM pea with a modified
glycosylation pattern which may result in an increased antigenicity and
adjuvanticity. When the experiment was repeated with the same mouse
strain by another laboratory, however, these findings of allergic
responses could not be confirmed.
The adjuvant effect of Cry1Ac on systemic and mucosal specific
antibody responses and on cellular immunity after intra-nasal or intra
peritoneal administration of microbial antigens was evidenced. It was
shown that after intra gastric administration Cry1Ac was a mucosal and
systemic adjuvant as potent as cholera toxin (CT), a major oral adjuvant.
It induced a Th2 type immune response, for the hepatitis B surface
antigen and bovine serum albumin; its effect depended on the antigen
and the route of administration.
However, adjuvanticity of Cry 1 Ab was not found to be based on the
same mode of action than that of CT and the capacity of Cry 1Ab to
enhance an allergic reaction to peanut allergen was not observed in
conditions of administration corresponding to the consumption of MON
810 maize very likely because the concentration of the newly expressed
protein Cry 1Ab in MON 810 is much lower than the doses used by
Vazquez-Padron to observe an adjuvant effect.
A mechanism of action involving macrophage activation has been
described by Moreno-Fierros in 2013 and Torres-Martínez in 2016.
Several other studies were performed on laboratory animals and they
concluded in most cases on the non-allergenicity (including sometimes
non-adjuvanticity) of the GM-feed products in the intended conditions of
use.
Some studies have been performed in livestock animals to evaluate the
safety of the GM feed, but most of them did not focus on the
allergenicity issue or even on alteration of the immune response.
Few of the studies that included immunological parameters were the
studies by Walsh and others in 2011 and Buzoianu and others in 2012.
In these studies, the authors assessed the effect of short-term feeding (31
days) of genetically modified Bt maize (MON810) on immune responses
and growth in pigs.
Pigs were fed a diet containing 38.9% GM or non-GM isogenic parent
line maize. Feeding GM maize to weanling pigs had no effect on growth
performance or body weight. They observed, however, effects of the
GM maize on immune responses. IL-12 and IFNγ production from
mitogenic stimulated peripheral blood mononuclear cells decreased after
31 days, as compared to the control non-GM diet. In these experiments
the pro-inflammatory cytokines IL-6 and IL-4 from isolated splenocytes
were also measured. Their production was increased in response to
feeding GM maize. However, the increase in production of antigen-
specific IgA and IgG antibodies that accompanies a Th2-mediated
response was not observed. Furthermore, the increase in cytokine
production, while statistically significant, was numerically small and
unlikely to be of biological relevance.
This indicates that feeding GM maize did not elicit an allergenic
response. This concluded that exposure to GM maize did induce some
alterations in localized and peripheral immune responses in weanling
pigs, which require further investigation. The lack of Cry1Ab specific
immunoglobulin production in plasma however suggests that the
immune response was not allergenic.
In further studies by Buzoianu pigs were fed MON810 maize diets for
maximum 110 days. They found no changes in the production of IL-6,
IL-4, IL-8 and TNFα from mitogen-stimulated or resting PBMC
between pigs fed Bt maize for the entire 110-day study period, in pigs
that were older when first fed Bt maize, or in control diet pigs. Also, no
Cry1Ab antigen-specific antibody response was detected in this study.
There were no indications of a Th 2-mediated allergic response to the
Cry1Ab toxin. This concluded that long-term feeding of Bt maize to pigs
did not elicit an allergic or inflammatory-type peripheral immune
response because of the lack of antigen-specific antibody production and
the absence of alterations in T cell populations and inflammatory
cytokine production.
A few multi-generational studies in livestock animals were performed
Trabalza –Marinucci conducted a longitudinal study on three
generations of sheep fed diets with Bt176 maize- or non-GM maize.
They evaluated the breeding performance, reproductive traits,
hematological parameters, antioxidant defenses, lymphocyte
proliferative capacity, phagocytosis and intracellular killing of
macrophages, and ruminal microbial population and immune response to
Salmonella abortus ovis vaccination for three years. They found no
adverse effect of the GM diet for these parameters. However, the authors
claimed they observed proliferative activation of basal cells of rumen
epithelium in all GM maize-fed ewes, as well as smaller cell nuclei in
hepatocytes and pancreatic acinar cells, which contained increased
amounts of heterochromatin and perichromatin granules in GM-fed
lambs.
They also reported that immune response to Salmonella vaccination was
more efficient in GM maize-fed sheep. They advised the performance of
new longitudinal studies with a special focus on the effects on the
immune system. Snell, however, raised major criticisms against these
results. First, it appears that they did not use an isogenic line as a non-
GM-comparator and both lines were not grown under similar conditions.
They did measure the composition of the GM and non-GM diets and
claimed that the compositional differences were “so minor that they are
unlikely to be of any biological significance”. However, this conclusion
is not supported by data. Secondly, there is no evidence that the
cytosolic differences are reproducible in independent replicates or if they
are observed at different time points.
Overall, the short-term, long-term and multigenerational studies mostly
show that the GM feed products described in this review were as safe as
conventional feed for livestock animals. No allergic responses were
reported, although this was often also not included in the parameters
analyzed.
There were some studies where safety concerns were raised by the
authors, and long-term investigations were recommended. Some of these
studies were performed, but they suffered from weaknesses in setup or
statistical analysis and were refuted by other scientists. In general,
information on immunological responses, and especially allergic
reactions, after feeding livestock animals GM products is very limited.
In conclusion, there are no case reports of allergic reaction or
immunotoxic effects resulting from the feeding of currently EU
approved GM feed products to livestock as compared with
corresponding non-GM feed. For future products, current guidance for
risk assessment (Codex Alimentarius, 2009, EFSA, 2011a, EFSA,
2011b, Commission Regulation (EU) No 503/2013 of 3, 2013) seem
appropriate, but could be refined on a case by case basis by the
following considerations which would also allow a better management
and prevention of the allergy risk.
Very little research focused on determining immune parameters of
livestock animals in GM feeding trials, and especially parameters
involved in allergic reactions. Most of the studies performed in livestock
focused on the assessment of nutritional parameters and performance of
livestock animals. Allergenicity and immunotoxicity testing is more
often performed in rodent models. It is, however, questionable whether
these models give a good representation of the allergenicity of proteins
for livestock species. It would thus be important to develop validated
and standardized animal models for the assessment of allergenicity in
farm animals.
Feed allergy is highly specific to individuals, both in the specific protein
that binds the IgE antibodies and in the minimum (threshold) dose of
allergen that elicits an allergic reaction. Minor feed allergens, that give
only a reaction in a small proportion of the population, will most likely
not be detected when studied in animal trials.
For second generation of GM plants, where novel proteins are
introduced or where the composition is changed for nutritional or health
purpose, post-market monitoring could be applied.
When technically possible, a PMM could involve screening of animals
for IgE-sensitization to the protein by in vitro tests or by skin prick
testing. Since specific IgE levels in blood are not always related to
allergy, careful evaluation of clinical findings (in first instance presence
of diarrhea, reduced growth performance, weight loss) and exclusion of
differential diagnoses should take place. A strategy of post-market
monitoring could focus on case-control studies in regard to specific age
groups (i.e. weaning, change of group composition, change of diets) or
extremes of production systems in different species (i.e. high production
pig systems, veal calves, etc.)
Additionally, to perform a post market monitoring program of potential
food allergies in livestock animals, there is a need for exchanges of clear
and reliable information between stakeholders with regards to the
introduction of new feed products derived from GMOs, their
identification and the adverse effects that their consumption might have
previously caused in livestock animals.
This involves farmers, animal breeders, health professionals, feed
industry, risk assessors and risk managers. Networks of these groups
such as professional and farmer’ organizations might serve as primary
contact point for dissemination and validation of pertinent information.
This would allow collecting accurate data on the actual exposure and
significantly and specifically relating any adverse effect to the intake of
the GM food in order to substantiate or rule out potential differences of
allergies to GM-versus non-GM- products.
Immune-mediated reactions have been observed in young farm animals
(calves, pigs) and in intensively reared fish species (salmon, trout) in the
context of replacing proteins of animal origin (e.g. from cow's milk or
fish) with cheaper proteins of vegetal origin (mainly soybean) in food
formulas.
They can be prevented by using food ingredients appropriately
processed using single or combined treatments, including heat, enzymes
and/or organic solvents. Additional work is still needed in fish and pets
but also on laboratory animals for understanding better the underlying
immune mechanisms involved in adverse reactions to feed and develop
refined strategies for management and prevention.
Under practical conditions, feed allergy (to most likely soy proteins)
may play a role in gastrointestinal disorders. This might especially be
the case in calves, and piglets after weaning. The early substitution of
artificial diets for mother's milk may enable the uptake of allergens,
leading to allergic manifestations and impairment of gastrointestinal
function. This will most likely occur for only a short period after which
weaned animals outgrow their soy allergy. Few studies actually report
feed allergy in domestic animals, which thus does not appear as a major
public health concern. However, adverse immune reactions or
intolerance to feed/feed components may occur at critical steps of
animal rearing (e.g. after weaning in pigs) or under particular conditions.
As noted by EFSA (2010), whilst animals and humans may share some
allergens in common, no database is known to provide comprehensive
information on compounds that would be specifically allergic to animals
or humans. Therefore, EFSA recommended that the human allergen
databases used for bioinformatics-supported assessment of newly
expressed proteins in GMOs are completed with allergens for animals.
There is no specific indication of allergies following a different
mechanism in animals than in humans, whilst the level of exposure and
digestive physiology may be different from those in humans. Test
animal models for allergies in pigs and other domestic animals should be
further developed to better understand underlying mechanisms of
allergic sensitization (and better assess and prevent the allergic risk) in
humans but also in those particular species of interest.
Similar considerations may also pertain to adjuvanticity which may be
considered in the assessment of certain GMOs, particularly in IR-GM
crops expressing Bt proteins such as Cry1Ac or, more generally, when
the newly expressed protein(s) functionally or structurally resembles a
known strong adjuvant.

A collection of genetically modified chicks.

A genetically modified cow.

 Horizontal gene transfer
In evolutionary biology few issues have received as much consideration
in the last 26 years as horizontal gene transfer (HGT, or lateral gene
transfer, LGT), with almost 11,000 papers among which article, review,
proceeding paper, book chapter and meeting abstract have appeared on
the topic since 1990 (10,704 papers, Thomson Reuters Web of Science
Core Collection December 2016).
Horizontal gene transfer (HGT) refers to the transfer of genetic material,
independent of reproduction, to a living cell or organism, followed by its
expression. It takes place extensively among prokaryotes, plays a key
role in the evolution of genomes and has been documented within and
between the Archaea, Bacteria, and Eukarya domains as well as viruses.
HGT can take place in soil, water, the gastrointestinal tract of humans
and animals, as well as in some food (e.g. milk). It can occur naturally or
be induced in vitro by chemical or physical conditions and may be
temporary and not persist in offspring.
Theoretically, all genes, including highly conserved genes such as
ribosomal genes, appear to be capable of HGT.
The following figure shows bacterial transformation, bacterial
transduction and bacterial conjugation respectively.
Each form of HGT involves different risk considerations; for genetic
engineering, these risks are commonly addressed through legislation. In
particular, in accordance with the Commission Regulation (EU)
503/2013 of 3 April 2013 on applications for authorization of genetically
modified food and feed, “the applicant shall assess the probability of
horizontal gene transfer from the product to humans, animals and
microorganisms and any potential associated risk when intact and
functional nucleic acid(s) remains in the genetically modified food and
feed”.
The capacity of microorganisms to acquire new genetic material differs
between and within species and may influence the potential for
horizontal gene transfer (OECD, 2010). HGT requires a number of steps
including introduction, uptake and expression of foreign genetic
material. The probability that a specific gene will be successfully
acquired by a new host depends on the specific mechanisms of HGT
(transformation, transduction, or conjugation), on the relationships of
these mechanisms to the types of nucleic acids (single-stranded, double-
stranded, linear, circular, etc) as well as environmental conditions.
Additionally, if the transferred DNA does not provide selective
advantage, it is likely to be lost in the population.
Various indirect forms of HGT have also been identified that involve a
“mediator” organism, which facilitates gene transfer from a host to the
final recipient organism. The most familiar is virus-mediated gene
transfer between bacteria.

.
Mechanisms and detecting patterns of HGT:
With the purpose of evaluating the impact of the transfer of GM material
between organisms, it is important to take in consideration naturally
occurring gene transfer and the mechanisms behind it.
Mobile Genetic Elements (MGEs), which are a dominant feature of most
genomes (e.g. they constitute 80% of the maize genome), are segments
of DNA that encode enzymes and other proteins that mediate the
movement of DNA within genomes or between bacterial cells. In terms
of HGT, they are one of the main conduits for HGT and play an
important role in the spread of antibiotic resistance genes and
pathogenicity determinants.
HGT is a two-step process. Initially, there is transfer of genetic material
across the cell membrane and other cover structures such as a cell wall
or nuclear membrane, followed by stable incorporation of the foreign
genetic material into the genome of the recipient organism. Each HGT
event may be accompanied by expression of the genetic material or
changes to expression of endogenous genes.
The most important HGT mechanisms described in the literature include
conjugation, natural transformation, and virus-mediated transduction.
Transfer by conjugation requires direct contact between donor and
recipient cells and is mediated by conjugative or non-conjugative
plasmids or transposons. The Agrobacterium conjugation system, by
which the Ti-plasmid is transferred into plants, is a model of inter-
kingdom DNA transfer (Gelvin, 2000) There are many examples of
inter-species and inter-genus transfers of DNA by conjugation in
food/feed and in the intestine.
The transformation mechanism of prokaryotes involves integration of
free extracellular DNA that becomes incorporated into the genome
(Lorenz and Wackernagel, 1994). Prokaryotic genomes are highly
dynamic, they are usually replete with HGT, undergoing continuous
gains, often from outside the species, genus, or family, and losses
through deletion.
To date, approximately 90 bacterial species have been found to be
naturally transformable, many of which are important with respect to the
food chain (e.g., Bacillus subtilis, Campylobacter sp.). Likewise,
different types of cells that can be cultured in laboratories can acquire
and integrate free DNA by transformation; competence in some bacterial
species (e.g. E. coli) can be induced in vitro by chemical or physical
conditions.
Transduction is the transfer of DNA from one prokaryotic cell to another
by bacteriophages, viruses that infect and replicate within bacteria.
Bacteriophages are ubiquitous in the environment and are also abundant
in the gastrointestinal tract of animals and humans.
Among the different mechanisms, the conjugation is the process of
major relevance for translocating DNA between bacteria. It can also
occur between remotely related species, even between members of
different domains of the prokaryotes, the Archaea and bacteria.
A combination of different approaches may be necessary to identify and
confirm HGT:
1)Phylogenetic analysis: is considered the most rigorous method for
detecting past HGT events;
2)Experimental evidence: involves the use of laboratory and field study
data to assess for HGT;
3)Nucleotide compositional analysis: provides a rapid method to assess
and detect gene alterations;
4)Inconsistent evolutionary scenario: involves detection of genetic
signals in distantly related organisms vs closely related organisms as a
means of determining possible HGT.
HGT from GM crops to animals:
One of the major concerns raised by the scientific community and
general public is regarding the possibility that DNA introduced into
genetically modified crops could be transferred to bacteria within the
gastrointestinal tract of livestock (EFSA, 2007, EFSA, 2009, EFSA,
2013), and in food products of animal origin.

This concern led to a considerable number of published studies on fate

of recombinant DNA as reported by a comprehensive review of
scientific literature. Higher organisms have developed sophisticated
defense mechanisms for inactivating foreign constituents.
The first line of host defense is the intestinal epithelial border, which is
not an absolute barrier to macromolecules, given that undigested
material can be transported across the intestinal epithelium to lymph and
blood and via the portal vein to the liver. In the blood, nucleases
hydrolyse DNA unless association with DNA-binding proteins can
protect it whilst in the liver, DNA is degraded by endothelial cells.
Failure of defense mechanisms could potentially expose host tissues and
organs to foreign DNA.
Gut microbiota hosts a variety of microorganisms inhabiting the
mammalian gastrointestinal tract. The composition of this microbial
community is host specific, evolves over time and is subject to both
endogenous and exogenous modifications. It is directly involved in
various aspects of physiology, from nutritional status to stress response.
The mechanisms through which microbiota exercises its influences are
not entirely clear, however, they include recognition of bacterial
epitopes by both mucosal immune cells and intestinal epithelial and
elaboration of signaling molecules which are part of a complex system
of communication that governs basic cellular activities and coordinates
cell actions.
To date, the competence of the bacteria present in the lower
gastrointestinal tract (GIT) to acquire dietary DNA by natural
transformation is currently not well understood, nevertheless, the
occurrence of horizontal gene transfer in the GIT remains a credible and
testable hypothesis.
Although it is difficult to provide a realistic estimate of DNA intake for
farm animal diets, it has been calculated that the DNA content of most
food crops is lower than 0.02%. For a 600 kg dairy cow, fed (dry matter
basis) with GM maize as ingredient, transgene DNA consumption would
amount to 2.6 mg per day, which represents 0.00042% of the total
dietary DNA intake (Beever and Kemp, 2000). Actual exposure is likely
lower due to the influence of various factors affecting dietary DNA
stability and persistence in food sources including food composition,
processing, and storage practices.
The fate of plant dietary DNA in the GIT and tissues has been
investigated in various animal feeding studies, including cattle, sheep,
pigs, poultry, rats, fish, and wild animals. Moreover, experimental
feeding studies and publications with controversial results have recently
been assessed by Van Eenennaam and Young (2014).
Low level of nuclear, recombinant, plant DNA fragments were identified
in different segments of the GIT, particularly in monogastric animals
(pigs and rats) and polygastric avian species (e.g., poultry). In contrast,
multicopy chloroplast genes were often detected in liver, kidney, spleen,
blood and muscle of broilers, pigs, and to a small extent, in ruminants.
Generally, based on studies performed with animals fed genetically
modified soy, maize and cottonseed, recombinant plant DNA has never
been detected in milk, eggs or blood, even when DNA fragment of
multicopy chloroplast genes could be detected.
As a singular event, small fragments of recombinant DNA were detected
in milk samples from the Italian market, which the authors interpreted as
an indicator of fecal or airborne contamination with feed particles.
HGT takes place mostly in the lower part of the GIT (e.g., the colon) due
to the abundance of nutrients as well as high bacterial density and
diversity. Extensive degradation of DNA in the gastrointestinal tract
occurs, reducing the size and frequency of DNA transfers.
In particular, Netherwood and others have reported evidence in 2004 of
low-frequency transfer of a small fragment (180 bp) derived from GM
soybean to bacteria within the small intestine of human ileostomists.
Nevertheless, only very low concentrations of the small DNA fragments
were detected in samples of microorganisms taken from the small bowel
of three of seven ileostomists. Moreover, the small fragments were
detected only after two steps of DNA amplification while, in feces from
human volunteers with intact digestive tracts, the gene could not be
detected indicating that the introduced gene is entirely degraded in the
large intestine.
Moreover, El-Sanhoty and others verified DNA transfer in rats fed GM
crops, through DNA extraction and purification from digesta, organs and
feces in 2006. This paper proves that DNA is moderately resistant to
mechanical, chemical and enzymatic activities within the rat GIT.
Nevertheless, this DNA did not lead to transgenic gene expression in
somatic cells or demonstrable integration into the germ line of the
animals.
HGT and antibiotic resistance:
From GM plants the only genes that are expected to be successfully
transferred to bacteria are other bacterial genes, including antibiotic
resistance genes employed in the transformation process.
Potential recipient bacteria for antibiotic resistance genes include
bacteria that infect plants or reside on plant surfaces, soil bacteria,
endosymbionts, and mammalian gastrointestinal bacteria that consume
plant products.
Antibiotic resistance genes are widespread in microbial populations.
Some of these resistance genes are enclosed on conjugative plasmids
and can be transferred across microbial populations, in particular where
bacteria have a competitive advantage. Likewise, food can act as a
vehicle for the transfer of antibiotic resistant bacteria and/or antibiotic
resistant genes to human in several ways.
The large majority of antibiotics are used worldwide in primary animal
production and in some measure, in plant production and apiculture;
antibiotics are also intentionally added during the food processing
(starter cultures, probiotics, bio conserving microorganisms and
bacteriophages) or through cross-contamination with antimicrobial
resistant bacteria during food processing. However, within the EU,
during animal production antibiotics are banned when used for growth
promotion and restricted to therapeutic measures.
Selectable marker genes and antibiotic resistance:
The utilization of antibiotic resistances marker genes (ARMGs) as a
selectable marker in cell transformation has been common practice in
plant genetic research. There are two ways in which antibiotic resistance
genes can end up in the genetically engineered plants: through molecular
cloning of the transformation vector in bacteria and/or via the trait-
conferring gene for identification/selection of successfully transformed
plant cells. In addition to antibiotic resistance genes, herbicide tolerance
genes and reporter genes coding for, e.g. metabolic traits, are used for
isolation of transformed plant cells.
Currently, a small percentage of the more recently developed
recombinant DNA plants projected for commercialization contain
antibiotic resistance genes. In Europe, measures to limit the use of
marker genes have been taken, even though the use of ARMGs has not
been completely prohibited. The Directive 2001/18/EC on the deliberate
release and placing on the market of GMOs states that” Member States
and the Commission shall ensure that GMOs which contain genes
expressing resistance to antibiotics in use for medical or veterinary
treatment are taken into particular consideration when carrying out an
environmental risk assessment, with a view to identifying and phasing
out antibiotic resistance markers in GMOs which may have adverse
effects on human health and the environment”.
To minimize this supposed risk, it has been proposed to use transgene
sequences lacking DNA similarity to bacterial sequences or to
incorporate introns into GM plant transgene sequences.
Recently, several strategies have been developed to remove marker
genes from GM products while preserving the transgenes of interest. A
review by Yau and Stewart (2013) describes the existing strategies for
marker genes removal and looks at possible future research directions.
Afterward, Breyer and colleagues presented the results of a three-step
analysis of selectable markers and reporter genes along with methods
aiming at developing marker-free GM plants. The efficiency,
practicability and biosafety of the various selection approaches is
discussed and considered taking into account their present use in light of
the long history of use of ARMGs in plant biotechnology.
Safety and risk assessment of HGT:
In 2000, the European Commission funded a thematic network titled
ENTRANSFOOD on the safety assessment of genetically modified food
crops. The working group (linked to GMOBILITY project “Safety
evaluation of horizontal gene transfer from genetically modified
organisms to the microflora of the food chain and human gut”) dealing
with HGT produced a comprehensive review based on published
scientific knowledge and on the results from the GMOBILITY project.
Assessing risks of HGT of recombinant DNA in foods derived from GM
crops involves estimating both the likelihood of transfer of DNA from
GM crops to microorganisms or human cells and the impact of such a
transfer event.
The uptake of DNA from GM crops by microorganisms may take place
at any step in the production or consumption of a GM crop including
cultivation, transportation and storage, processing, consumption,
digestion, and following excretion.
For example, transformation during the production of silage from GM
plants would affect bacteria that enter the intestines of cattle. As a result,
the acquired DNA may spread throughout the intestinal flora by
different HGT mechanisms.
HGT from GM crop plants to a recipient bacterium is thought only to
occur by homologous recombination. Adjacent genes flanking the region
of homology may be included where the homologous region is present in
a circular molecule or where multiple regions of homology are present.
Expression of the acquired genes in the recipient cell is a prerequisite for
both beneficial and adverse effects of gene transfer but, microbial, plant,
and animal cells have quite different expression systems. Consequently,
genes transferred from one cell to another are frequently not expressed at
all or only at very low levels. Moreover, based on a model for genome
evolution developed by Vogan and Higgs (2011), natural selection leads
to gradual improvement of the replication accuracy and gradual decrease
in the optimal rate of HGT.
The assessment of risks from HGT usually focuses on genes, e.g.,
antibiotic resistance genes, that may confer a selective advantage to
microbial cells. The presence of antibiotics and antibiotic usage in
different contexts are key factors in driving the dissemination and the
selection of antibiotic resistance genes. The biosafety of the use of
ARMGs has been reviewed by several authors such as Bennett, Gay and
Gillespie, Goldstein , Nielsen and Ramessar.
A lot of attention has been focused on the transfer of marker genes from
transgenic plants to soil and plant microorganisms as this may pose a
risk to human or animal health by compromising the therapeutic value of
relevant antibiotics (WHO, 2007, EFSA, 2009).
In particular, GMO and BIOHAZ Panels (EFSA, 2009) came to the
following conclusion:
•Antibiotic resistance traits included in GM plants or their derived
products are evaluated on a case-by-case basis with regard to their safety
for humans, animals and the environment by the EFSA GMO Panel
according to the principles expressed by Directive 2001/18/EC of the
European Parliament and based on the updated EFSA guidelines.
•The transfer of antibiotic resistance marker genes from GM plants to
bacteria has not been shown to take place either in the laboratory or in
natural conditions in the absence of sequence identity in the receiver
bacterial cell.
•DNA transfer from GM plants to bacteria, when occurring, is
considered to be of low frequency compared with gene transfer between
bacteria.
•Current metagenomic analyses of total bacterial populations have
shown that resistance determinants of kanamycin, neomycin and
streptomycin (marker genes that are present in GM plants for which an
application has been submitted to EFSA) are present in all environments
considered. Such resistance genes may be selected from this
environmental reservoir and spread among bacteria. There are
limitations related to sampling, detection, evaluation of exposure levels
and the incapability to assign transferable resistance genes to a definite
source.
•Despite these uncertainties, currently the adverse effects on human
health and the environment resultant from the transfer of these antibiotic
resistance genes from GM plants to bacteria are unlikely.
In the year 2004, Guy van den Eede stated that the use of specific
antibiotic resistance genes contained in GM crops should be evaluated
on a case-by–case basis considering three major factors: the likelihood
of transfer of an antibiotic resistance gene from a transgenic plant to a
bacterium; the frequency of occurrence of the resistance gene in
microbial populations; and the importance of the antibiotic, and the
extent of clinical and veterinary use. On the basis of frequency of
occurrence in microbial populations and importance of clinical use of
the relevant antibiotics, the antibiotic resistance genes have been
assigned to three risk groups.This approach has also been adopted by the
EFSA scientific panels on GMOs and biohazards.
•Group I contains antibiotic resistance genes that are extensively
distributed among soil and enteric bacteria and confer resistance to
antibiotics that have no or only minor therapeutic relevance in human
and veterinary medicine; (e.g. nptII gene conferring resistance to
neomycin and kanamycin);
•Group II contains antibiotic resistance genes that are broadly distributed
in microorganisms and confer resistance to antibiotics that are used for
therapy in distinct areas of veterinary and human medicine (e.g., bla
gene conferring resistance to ampicillin);
•Group III contains antibiotic resistance genes that confer resistance to
antibiotics significant for human therapy and therefore should be
avoided in the genome of transgenic plants (e.g., npt III gene conferring
resistance to amikacin). For example, the EFSA Scientific Panels do not
recommend the usage of any ARMGs encoding resistance to
tetracyclines (group III) whereas recognize the npt II ARMG conferring
resistance to kanamycin (group I) as being safe (EFSA, 2004, EFSA,
2007, EFSA, 2009).
Ku and Martin have (Ku and Martin, 2016) developed an approach to
summarize the effects of HGT in prokaryotic and eukaryotic genome
evolution. According to these authors, the occurrence in sequenced
eukaryotic genomes, of a DNA sequence that codes for a homologue of
a prokaryotic proteins with minimally 70% amino acid identity is most
likely a contamination artifact rather than a laterally acquired gene.
Their findings indicate that eukaryotes do not acquire genes through
continual LGT like prokaryotes do. Major gene acquisitions do occur in
eukaryote evolution, but these correspond to endosymbiotic events.
Transfer of GM traits to microorganisms by transformation is a limited
possibility and is only significant if the new trait is expressed and
confers selective advantage.
Kleter evaluated the function, characteristics, and potential health impact
of the introduction of different transgenes of microbial origin into
commercial GM plants in 2005. The authors concluded that unintended
horizontal gene transfer to bacteria was unlikely to raise health concerns.
Whereas uptake of ingested recombinant (foreign) DNA by mammalian
somatic cells has been demonstrated, there is no evidence that ‘ingested’
DNA will end up in germline cells.
Transfer of antibiotic resistance marker genes from GM plant to the gut
microflora of humans and animals and their expression is most probably
a rare event, given the low amounts ingested and degradative conditions
in the gastro-intestinal tract, however in Europe, measures to limit the
use of marker genes have been taken (EFSA, 2009, EFSA, 2011a; EC
2013).
These considerations are generally consistent with the conclusions of the
latest EFSA External Report (EFSA, 2013), which reviewed current
scientific literature focusing on the safety assessment of GM food and
feed which have substantial modifications in composition, metabolism
and/or physiology of the plant (“novel” traits). According to the report, a
precise prediction of the fate of dietary DNA in mammalian cells is
currently unachievable in the majority of studies and for this reason
should not form the basis of risk assessment for novel GM food and
feed. It is also reasonable to suggest that the risks of transfer of
transgenic DNA are equal to that for non-transgenic DNA.
 Lower Mycotoxin Levels and GM crops
The debate on the advantage of using Bt GM crops (i.e. expressing
insecticidal toxins from Bacillus thuringensis), for their capacity to
control mycotoxin content is still open and needs to be further supported
with evidence of the potential benefits of the mitigation effects to be
presented to the stakeholder community. The topic is of particular
interest as the impact of mycotoxins on farm animals is an important
issue and mycotoxin reduction is crucial for animal health, productivity
and farm economy. All GM maize events (mainly Bt maize) authorized
for import, processing, and cultivation and commercially available in the
EU market, were produced for management of insect infestation but not
for fungal or mycotoxin control. However, insect-resistant GM maize
crops show evidence of reduction of fumonisins contamination as an
advantageous side effect with potential yet unproven positive
consequences of the derived feed on animal health. For other Fusarium
toxins and aflatoxins, however, the evidence of possible control remains
inconclusive.
This review aims to scrutinize the mycotoxin scenario in Bt GM crops
and explores the opportunity of using a biomarker of mycotoxin
exposure to monitor the effect of Bt GM/non-GM feeding on animal
performance. With this, it could be verified if any kind of positive side
effect might be attributed to lower level of mycotoxins under a Bt GM
crop-derived feed-based feeding regime.
Mycotoxins and GM crops:
It is widely reported that mycotoxin content in Bt GM crops (especially
Bt maize) is reduced by virtue of its increased resistance to pests’ attack
and of the higher integrity of the GM plant providing fewer plant
wounds that could act as points of entry for secondary infections by
mycotoxin-forming molds. In fact, the inserted genes coding for Bt
proteins, such as Cry1Ab and Cry1Ac, confer crop protection to
Lepidoptera (e.g. Ostrinia nubilalis and Sesamia nonagrioides) and/or
Coleoptera (e.g. Diabrotica virgifera) pest attack and make the plant less
prone to plant or kernel damages.

A mycotoxin produced by fungi.

Mycotoxin development on plants is favored by the interaction of
several factors: climate conditions (such as temperature and humidity,
heating and drought stress), agricultural practices (such as crop rotation,
tillage practices) or hybrid choices. Moreover, pests and diseases play a
crucial role in the fungal attack and infestation since insect wounds and
damages on plant and kernels facilitate and increase the spore migration
and colonization, especially in case of Fusarium ssp. (CAST report,
2003). Furthermore, the mycotoxin production and accumulation, which
starts in the field, persists and may continue during either harvesting or
storage representing a concern for human and animal health because of
their toxicity (Wicklow, 1994).
Numerous experimental field trials of Bt maize have been conducted
worldwide; a list of the most relevant field trials with the GM event used
and the mycotoxin assessed is reported in Supplementary Material Table
S1. Studies carried out in Germany compared mycotoxin levels in GM
and isogenic maize lines. The only strong relationship highlighted is
between insect pest damage and fumonisin levels in Bt GM crops, being
recognized that Bt hybrids may exert a secondary effect of mitigating the
fungal plant attack and consequently, Bt maize that shoes a lower pest
attack and lower fungal infection, also show lower fumonisins levels
when compared with isolines. Among fungus that very often attack
maize, F. moniliforme and F. proliferatum, use wounds as the preferable
ports of entry for infection and show a positive correlation among the
GM plant trait resistance to the pest disease, the level of fungal infection
and the mycotoxins presence in field trials.
However, summarizing all the outputs of GM and isolines comparison,
the efficacy of fumonisins reduction decreases when:
•GM hybrids do not express the Cry1Ab protein in kernels;
•Other insect pests occur, for example western bean cutworms, which
are not controlled by the Cry1Ab protein.
•Extreme environmental conditions or abiotic plant stress limit the effect
of GM traits since the strong pressure of the adverse climatic conditions
(such as excessively hot and dry conditions), may over-stress fumonisin
B1 production in maize.
•The comparison of Bt hybrids is done with conventional hybrids
expressing a natural resistance to Fusarium ssp., which equals the Bt
protection effect.
Despite the known effect of Bt maize on fumonisins content in the field,
still little is known regarding the accumulation of mycotoxins in Bt
maize during the post-harvest stage. Recently Rocha in 2016
investigated the levels of fumonisins and the FUM gene biosynthesis
cluster expression on Bt and non Bt maize, inoculated with F.
verticillioides, post-harvest, during the different periods of incubation
(10, 20 and 30 days). According to this study the fumonisin B1
production was significantly lower in Bt hybrids (P < 0.05) while no
statistical difference for fumonisin B2 production (P > 0.05) was
observed. The FUM genes were expressed by F. verticillioides in all of
the samples, and it was found a lower expression of a number of FUM
genes and a weak association between fumonisin B1 production and the
relative expression of some of the FUM genes in the Bt hybrids.
As regards other mycotoxins of concern that may also occur and/or co-
occur in maize (e.g. as aflatoxins, deoxynivalenol and zearalenone) the
field test comparisons have never highlighted any significant correlation.
Experimental field trials in Germany showed mixed results for the
deoxynivalenol level registered. In United States, lower contamination
of aflatoxins were found but some field trials showed in some years,
higher aflatoxins concentration in Bt hybrids compared to conventional
hybrids. Also, aflatoxin levels where sometimes higher in Bt maize
hybrids, while mostly Bt hybrids showed lower aflatoxin levels in
comparison with non Bt isolines in those years with very high levels of
aflatoxins. The levels of aflatoxin accumulation and ear damage were
investigated in ten maize germplasm lines (including some selected for
resistance to aflatoxin accumulation) that were crossed to transgenic
maize expressing the Cry1Ab protein and non-transgenic; the obtained
results indicated that ear damage and aflatoxin accumulation was
significantly less in Bt than non Bt test crosses. Other field studies in
France and Spain demonstrated that Bt maize had lower mycotoxin
contamination in general, but the studies showed no significant or mixed
effect in reducing deoxynivalenol and aflatoxins. These contradictory
results may be explained by the fact that different mycotoxigenic fungi,
which may share temperature conditions for growth, spore release and
germination, may have different route of infection.
So, F. graminearum and Aspergillus flavus (deoxynivalenol and
aflatoxins producers, respectively) that infect the maize also through the
silks, do not experience any disadvantages on the Bt pest resistant crops,
while F. moniliforme and proliferatum (fumonisin producers), that
penetrate preferentially through the kernels, experience a disadvantage
for the integrity of kernels with a direct effect on the decreased level of
fumonisins. As a net result, it may happen that symptomless infected
kernels show considerable contamination levels of deoxynivalenol and
aflatoxins.
On the inconclusive results of the lack of protection against aflatoxins
contamination by Bt maize, Abbas and others reviewed a consistent
number of studies on the comparison of Bt and non Bt crops worldwide.
They also summarized the results of a set of experimental field trials
conducted in Southern United States in three different frames (1998–
2000, 2006 and 2008–2009) to compare fumonisins and aflatoxins levels
in non Bt maize hybrids and Bt hybrids. The investigated crop showed
variation of aflatoxin and fumonisin levels but the contamination of
fumonisin was significantly lower in Bt maize only in 1998 field studies
when levels of fumonisin were lower compared with the other two years
suggesting that Bt maize may be effective only under conditions of low
Fusarium infection. On the other hand, aflatoxin was lower in Bt maize
in 2006 when similar level of Aspergillus colonized the kernels of both
hybrids. In all the other crops, any significance evidenced a lower
content in Bt maize especially when the contamination levels were high
(>20 μg/kg) entailing that under conditions of high disease pressure the
contribution of the Bt gene was not able to reduce mycotoxins.
As regards the altered competition occurring between fungi in attacking
maize grains, the control on pests performed by GM event may reduce
the proliferation of some fungal species and favor the spread of other
strains, leading the presence of one mycotoxin in favor of another.
Folcher hypothesized in 2010 that the control of insects limited the
infection potential of F. verticillioides or F. proliferatum, an opportunist
fungus, favoring the development and the activity of F. graminearum,
which infested the plant and led to the production of deoxynivalenol
sometimes at higher levels in Bt varieties.
To verify or falsify this hypothesis, and to explore also other fungi that
populate the soil (e.g. Aspergillus) further work should be done taking
into account that the competition among these fungi can have a distinct
effect on the eventual levels of infection and mycotoxin contamination
in GM crops.
 GM stacked events
GM plants encoding for single Vegetable insecticide protein (Vip) have
limited spectrum of insect control when compared with “stacked”
transgenic maize events, which contain genes for more than one Bt toxin
widening the spectrum of insect resistance action.
Most of the new GM maize varieties in the EU market, are stacked GM
events and contain a combination of genes expressing more than one Cry
protein. For example, Maize DAS1507xDAS59122 expresses cry1F
gene, which protects against certain lepidopteron pests, cry34Ab1 and
cry35Ab1 genes, which provide protection to other coleopteran pests,
and pat gene, which confers tolerance to glufosinate-ammonium-based
herbicides. So, stacked events, expressing multiple Cry proteins, could
be more efficient against the fungal attack and with mycotoxin control.
According to Bowers in 2014 transgenically expressed Bt proteins being
active against multiple lepidopteran pests can provide broad, consistent
reductions in the fumonisin contamination. Fumonisin levels among non
Bt maize hybrids and Bt hybrids with transgenic protection against
European Corn Borer (MON 810) and Western Bean Cutworm
(TC1507) were compared in field trials conducted from 2007 to 2010 in
USA. According to the obtained results, Bt hybrids experienced less
insect injury, lower Fusarium ear rot occurrence, and fumonisin
contamination compared to non Bt hybrids. Moreover, under Western
Bean Cutworm infestation, Cry1F hybrids mitigated this risk of
fumonisin presence more consistently than Cry1Ab or non Bt hybrids.
Thus, transgenically expressed Bt proteins active against multiple
lepidopteran pests can provide broad, consistent reductions in the risk of
fumonisin contamination.
Up to now, very few studies have been performed to evaluate the
capability of Bt stacked events on fungal and mycotoxin control. Abbas
and coworkers conducted a study that compared ten commercially
available corn hybrids to evaluate the effects of the three genetic
backgrounds (stacked gene hybrids with multiple Bt genes; Round-up
Ready, RR2; and non-transgenic) on aflatoxin and fumonisin levels in
seeds. The herbicide tolerant events were stacked with transformation
event TC1507 expressing Cry 1F protein (two hybrids) and with
transformation event MON88017 x MON89034 expressing Cry1A.105,
Cry2 Ab2 and Cry3 Bb proteins (two hybrids). The experiment was
carried out in USA during 2011 and 2012, no significant differences in
mycotoxin production were found amongst the hybrid classes. In a field
experiment Brewer and others showed that a GM maize with multi-
stacked Bt genes (new stacked Bt VT3 Pro expressing Cry1A.105-
Cry2Ab and Cry3Bb proteins), compared with the non Bt hybrid isoline
under different water stress and insect pressure conditions, improved
ear-feeding worm control and crop yield and did not reduce the risk of
aflatoxin production. These data confirm that the route of infection of
aflatoxigenic fungi affects the effectiveness of Bt hybrids in reducing
aflatoxin contamination.
Anyway, GM crops have recognized efficacy in pests’ reduction but the
general effect of reducing mycotoxin levels cannot be converted in a
systematic result as the pest attack and insect damage is only one among
the different interacting factors that contribute to the mycotoxins
production. This means that the evidence cannot be oversimplified on a
statement that a GM crop reduces mycotoxin contamination as it is
sometimes claimed by shallow information, and that the secondary
agricultural benefit of reducing fungi and mycotoxins may be transferred
to the food and feed sector only if a holistic approach for mycotoxin
control is performed in the whole food and feed chain.
Mycotoxin animal health risk:
Animals are susceptible to exposure to the main mycotoxins (aflatoxins,
trichotecenes, zearalenone, ochratoxin A) via contaminated feed and
show different sensitivity, which varies depending on the animal species.
Monogastric animals are generally more sensitive to the toxic effect and
more susceptible to mycotoxicosesthan ruminants, and, among
monogastrics, pigs are considered the most sensitive farm animals.
Farm animals are constantly exposed to a mixture of mycotoxins from
the consumption of contaminated cereals and oilseeds that constitute
their diet. Table 2summarizes the most important toxic effects and
clinical signs that have been highlighted from scientific literature. Feed
blends with Fusarium contaminated grains seriously affects also
livestock performances leading to decreased feed consumption and
conversion, and decreased production (e.g. milk in dairy cattle, eggs,
meat); moreover, changes in metabolic, hematological and neurological
parameters have also often been reported.
Since maize is the main component in the feeding formulas, aflatoxins
and Fusarium toxins are the major mycotoxins that may occur and co-
occur in feed and thus of concern for animal health.
Aflatoxin B1 exerts acute toxicity in various animal species with liver as
the principal target organ (Eaton and Groopman, 1994, EFSA, 2004).
Thanks to its degradation by the forestomach flora, bovine species are
less sensitive compared to non-ruminants. Animals exposed to aflatoxins
may show clinical signs consisting of anorexia, icterus, depression,
weight loss, nasal discharge, gastrointestinal affections, haemorrhages,
ascites and pulmonary edema (EFSA, 2004). The liver metabolism of
aflatoxin B1 results in aflatoxin M1 that is excreted in milk (EFSA,
2004). The carry-over of aflatoxin B1 in milk as aflatoxin M1 has been
examined demonstrating that in dairy cows (milked twice daily) was
usually 1%–2% of the ingested AFB1 for low-yielding cows (<30 kg
milk yield/day) and up to ∼6% for high-yielding cows (>30 kg milk
yield/day).
Aflatoxin M1 can vary in individual animals being influenced by various
factors, such as the feeding regime, health status and individual
biotransformation capacity, and milk production (EFSA, 2004). Thus, in
this context, aflatoxin M1 in dairy cattle may be used as a specific
biomarker to be monitored to ascertain the exposure to aflatoxins
through contaminated feed.
As for Fusarium toxins, fumonisin B1 occurs in maize almost
constantly. It is hepatotoxic and nephrotoxic in all animal species tested
(IARC, 2002). Fumonisins are poorly absorbed and are rapidly excreted;
they are not metabolized in animal systems and the adsorbed fractions
are rapidly distributed and eliminated (IARC, 2002). The lowest
observed adverse effect levels (LOAEL) for fumonisin B1 have been
observed in horses and pigs, the most sensitive species (EFSA, 2005).
The acute toxicity of fumonisin is low, however, and it is known to be
the cause of two serious health problems in animals related with
disturbed sphingolipid metabolism: equineleukoencephalomalacia and
porcine pulmonary edema syndrome (IARC, 2002). Fumonisin B1
inhibits cellular sphingosine (sphinganine) N-acetyltransferase and this
inhibition results in an accumulation of sphinganine (Sa), sometimes
also sphingosine (So), and a depletion of complex sphingolipids in
eukaryotic cells (EFSA, 2005). So, the Sa/So ratio is a specific indicator
that can be monitored to ascertain the exposure to fumonisins through
the contaminated feed and it may also serve as a biomarker for the onset
of adverse effects.
Numerous countries worldwide have set maximum and/or guidance
levels to control mycotoxins in animal feed. The exposure to high levels
produces acute toxic effects leading to serious animal mycotoxicoses
and economical losses; however, prolonged exposure to low levels,
which are common in the feed, may also produce chronic toxic effect to
animals resulting in lower performances (Pettersen, 2012). In Europe,
aflatoxin B1 maximum level in feed material, complementary and
complete feed is set by the Commission Regulation (EU) No 574/2011.
The Regulation defines different maximum levels depending on the
animal species taking into account the specific sensitivities. As a matter
of fact, aflatoxin B1 is the only mycotoxin routinely controlled by the
feed chain stakeholders (e.g. farmers) because of the pressure of official
control, while, despite the EU recommended guidance values
(Commission Recommendation, 2006/576/EC), ochratoxin A and
Fusarium toxins are less controlled and monitored only when an adverse
clinical sign is recorded (e.g. food refusal, poor feed conversion and/or
weight gain).
Dersjant-Li reviewed the impact of low concentration of aflatoxins,
deoxynivalenol, and fumonisins on the performances of growing in pigs
and poultry; they estimated that each mg/kg increase of aflatoxin B1
decreased the growth rate by 16% for pigs and 5% for broilers; each
mg/kg increase of deoxynivalenol decreased the growth rate by 8% for
pigs and no response to concentration below 16 mg/kg for broilers; each
mg/kg increase of fumonisin decreased the growth rate by 0.4% for pigs
while no significant effect was observed for broilers. In conclusion, the
concentration that causes a 5% reduction in growth rate of the pigs and
broilers was estimated at: 0.3 and 1 mg/kg of aflatoxins, 21 and 251
mg/kg of fumonisins and 1.8 and 0.6 mg/kg of deoxynivalenol, for pigs
and poultry, respectively. To note that these levels are far above the EU
maximum level of aflatoxin B1 (i.e. 0.005 mg/kg for dairy cattle and
calves in complementary and complete feeding stuffs) and above the
guidance maximum values of fumonisins (i.e. 5 mg/kg for pigs and 20
mg/kg for poultry in complementary and complete feeding stuffs) and
deoxyinivalenol (0.9 mg/kg for pigs and 5 mg/kg for poultry in
complementary and complete feeding stuffs).
It is worth to mention that more often a simultaneous presence of
different mycotoxins in raw material could occur. The co-occurrence of
a number of mycotoxins is very common, especially in maize, which is
the most abundant ingredient in feed ration and thus the riskiest cereal
crop for the occurrence of different mycotoxins. The resulting toxic
effect of mixtures of mycotoxins has been scarcely studied and its nature
(if synergistic or additive) in animals or humans is a recent subject
matter.
GM and Mycotoxins in Feed:
Maize (whole plant, maize stover, cobs) and maize by-products (maize
oil, maize gluten, germ meal and germ) are fixed components of
livestock diets. Worldwide survey data indicate that maize is
unavoidably contaminated by fumonisins and that is very difficult to
find uncontaminated maize (EFSA, 2005). Thus, a beneficial reduction
of mycotoxins content experienced in the agricultural sector because of
GM crops should result in a net advantage in the GM feed chain:
unfortunately, these cannot be found or deduced from the scientific
papers.
The majority of the studies on the topic of GM/non GM feeding regimes
are focused to determine the differences in nutritional aspects and
animal performances (digestibility, feed intake, health and performances
of target animal species) and not to ascertain the specific effect induced
by the decreased percentage of mycotoxins (any kind) in the GM
feeding. However, few studies can be taken into consideration as they
compared GM and non-GM feeding regimes and reported findings of
mycotoxin contamination levels in feed. Delgado and Wolt (2010), for
example calculated long-term exposure and toxicological adverse effects
of fumonisin B1 in nursery swine productivity.
Taking into consideration diets containing GM Bt maize, conventional
non Bt maize, Bt and non Bt distillers’ dried grains with soluble
(DDGS), single and blended, fumonisin B1 was two times higher in non
Bt compared with Bt Maize, and diets where the corn fraction was
entirely from Bt corn showed the smallest fumonisin B1 exposure,
exceeding a defined chronic level of concern in 35% of occasions.
Piva and others attributed the weight gain of piglets and broilers GM fed
to the lower mycotoxin levels found in GM feed in comparison with the
near isogenic variety.
More in general, with the purpose to assess all sorts of health effects that
could arise from GM based diets, once verified the presence of
mycotoxins (and also pesticides) in feed, the majority of authors used
additives (in both GM and non GM based diets) to bind mycotoxins and
exclude any kind of confounding effect. In the study carried out by
Walsh in 2011, organic mycotoxins adsorbent (Mycosorb) was used in
all experiments nullifying any kind of consideration of possible
mycotoxin's side effect. On a transgenerational study for the evaluation
of effects of growth and health to nulliparous sows and offspring GM
maize fed, since mycotoxin were present (isogenic line having higher
levels of deoxynivalenol and zearalenone in comparison with Bt maize),
mycotoxins adsorbent and mold inhibitor were added to all the complete
diets as a preventive measure. Offspring of sows fed Bt maize improved
growth throughout their productive life compared to offspring of sows
fed isogenic maize.
The post-market environmental monitoring annual reports (published by
Monsanto) on the monitoring of Bt maize containing event MON 810
cultivated in various EU member states, which includes reports and farm
questionnaires, reported that farmers from Czech Republic and Slovakia
attributed the best performances of farm animals to the lower incidence
of mycotoxins in feed (BioMath GmbH, 2011 and 2012). This statement
cannot be verified due to lack of data on the actual mycotoxin levels in
the feeds used by these farmers and it would be important to further
considerations acquire other information about the presence and levels
of mycotoxins.
In conclusion, it seems very difficult to assert that animal performances
vary solely because of GM feed diets, in fact performances also depend
on a large number of other factors including feed contaminants among
which mycotoxins are of critical importance. At present, despite the
large use and experience on GM feeding, no specific data collection on
GM feed exposure has ever been carried out for this purpose, neither it is
possible to derive that the lesser content of mycotoxins in GM based
feed can be responsible of improvement of performances or health.
Therefore, an observatory at EU level with the purpose of following the
amounts of GM feed used, the mycotoxin levels and the animal
performances would be very helpful. On this respect, the use of animal
exposure marker for a number of mycotoxins (sphigosine/sphinganine
ratio for fumonisins or aflatoxin M1 in milk as a biomarker of aflatoxin
exposure) would help to monitor the mycotoxin exposure extent and to
deduce the extent of the mycotoxin's presence in feed. Taking into
account that animals follow a fixed diet, it doesn't seem a difficult task.
Moreover, to highlight that mycotoxin source in the feed chain may arise
from a large assortment of raw materials such as: cereal components (not
only maize but also oat, rye or wheat bran, for example), oilseed by
products (i.e. cottonseed meal, groundnut meal, palm kernel meal or
soybean meal), leguminous seed component (i.e. beans, peas or
cassava), silages (i.e. grass or straw), and pasture (grass, rye grass or
lupine) (Pettersen, 2012). The feed raw material and their associated
mycotoxin are summarized in Table 3, where for each crop the GM trait
and mycotoxins are reported.
As all these different crops are susceptible of mycotoxin contamination
and often feedstuffs are complex blends, different mycotoxins may
occur, and combined effects and increased adverse health effects may be
expected.However, other GM ingredients such as cotton, canola, linen or
sugar beet may be present in feed formulas in lesser amount. These GM
derived ingredients, all authorized products in the EU market, were not
included in the review as they are less important source either for
mycotoxins contamination extent or for the contribution as ingredient in
feed.
All GM maize events (mainly Bt maize) authorized for import,
processing, and cultivation and commercially available in the EU
market, were not produced for the purpose of fungal or mycotoxin
control. A lower mycotoxin contamination of maize and derived
products (i.e. feed) in Bt maize can therefore be a side effect to be
considered case-by-case for its impact along the processing food and
feed chain.
Scientific literature agrees in reporting that fumonisins contamination in
Bt maize decreases in comparison with the levels in the isolines grown
under the same conditions. From the evidence collected, it becomes
clear that GM crops and their use in the feed chain show a reduction in
fumonisins as an advantageous side effect with important returns in
terms of feed safety. Nonetheless, the lower contamination incidence
does not guarantee that the levels are under the maximum levels set by
the EU Regulation in maize or maize derived products, including feed
(Regulation 1881/2006 and 574/2011).
Field trials that studied aflatoxins, zearalenone, deoxynivalenol
contamination levels in GM and non-GM crops showed inconsistent
mixed results confirming that GM lines do not exert a systematic control
on the contamination of crops by these mycotoxins.
 Working of bt maize

Concluding, mycotoxins are a concern for animal health for their toxic
effect and their control in feed is priority to guarantee acceptable animal
performances. GM feed is susceptible to mycotoxin contamination as
much as non-GM crops even if lower content of fumonisin (in different
and specific conditions) has been registered, thus it is not possible to
identify distinctive positive health effect to be associated to the
consumption of GM feed related to mycotoxins.
While a post market monitoring (PMM) is asked to the applicant when
uncertainties about the pre market risk assessment are still unsolved, in
the case of mycotoxin reduction or control, considered only a side effect
of the genetically modification, a monitoring could help to understand
the extent of the benefit of this side effect.
For this purpose, a pan-European collection of data on mycotoxin
occurrence in feed, animal exposure and animal performances should
enable a better understanding of the effectiveness of the GM feeding
advantage as regards mycotoxin contamination. In this context, it would
be useful also:
i) To monitor aflatoxin B1 in GM crop-derived feed (a Maximum Level
exist for aflatoxin B1 in feed and maize) in combination with the
recording of animal characteristics or performances (weight gain, for
example);
ii) To collect data from official control;
iii) Gather and collect this information in a comprehensive data-base.
Moreover, since a Maximum Level also exist for aflatoxin M1 in milk
(Commission Regulation (EU) No 1881/2006) the routine measurement
of this toxin in milk in combination with the recording of milk
production rates in dairy cattle, can produce further useful information to
feed into a dataset with valuable data for post market monitoring
purposes. If, after scrutinizing a large extent of data, a sound relationship
between mycotoxin reduction and GM feed comes out soundly, all
stakeholders and farmers would be encouraged thanks to the
improvement of the safety of the feeds.
 Nutritionally Altered GM Crops

To date, most GM crops on the market are first-generation crops, which

involve enhanced agronomic traits such as insect resistance (Bt) and
herbicide tolerance (Ht) to improve crop production. First-generation
GM crops are considered substantially equivalent to conventional, non-
GM lines; the DNA insert that leads to synthesis of a gene product in
these plants does not interfere with the overall metabolism of the plant
cell. With regard to their nutritive value, the majority of animal feeding
studies have not found significant differences in production and health
parameters of animals that have consumed first-generation crops in
comparison to conventional, non-GM crops.

In fact, only one out of more than one hundred studies found a
difference in animal performance associated with first-generation GM
plants (EFSA, 2008), as a result of the lower level of mycotoxin in Bt
maize.
A recent extensive search of peer-reviewed literature and field
observations did not reveal unfavorable or perturbed trends in livestock
health and productivity by feeding diets containing first-generation GM
crop products. Generally, soybean is the most important animal feed in
the European Union (EU). Soybean and other commodity crops such as
maize, oilseed rape (canola) and cotton, have been genetically modified
for agronomic input traits, such as insect resistance and/or herbicide
tolerance (first-generation GM products).
These plants are both used in monogastric and ruminant diets as energy
and protein source. They are used as fresh or ensiled whole crop forage
(i.e. maize and lucerne), as a specific crop component (i.e. maize grain),
or as co-products (i.e. oilseed meals or maize stover). In 2015, twenty-
eight countries planted 179.7 million hectares of GM crops worldwide,
but most of these crops were grown in just five countries: the United
States, Brazil, Argentina, India and Canada (ISAAA, 2015). From these
countries, GM crops are exported to Europe, whereas the latter is quite
self-sufficient when it comes to maize. Most of Europe's maize
production is used in animal feed. Maize is the only GM crop cultivated
in the EU with 107,749 ha sown in Spain in 2015 and the remainder
(9121 ha) in Czech Republic (997ha), Romania (2.5ha), Portugal
(8,017ha), and Slovakia (104ha).
Before a genetically modified organism (GMO) can be marketed or
grown in the EU, it must be authorized under Regulation (EC)
1829/2003 (the 'GM Food and Feed Regulation') amended by the
implementing Regulation (EC) 503/2013. The authorization procedure
includes a scientific assessment by the Panel on Genetically Modified
Organisms of the European Food Safety Authority (EFSA). The EFSA
Panel assesses the safety of the GMO and the food or feed derived from
it.
The safety assessment follows a comparative approach, i.e. the food and
feed are compared with their non-GM counterparts in order to identify
differences reflecting intended or unintended effects of the genetic
modification which subsequently are assessed with respect to their
potential impact on the environment, safety for humans and animals,
(EFSA, 2011a). A scientific opinion is expressed by the Panel which is
considered by the risk manager (e.g. the Commission and member states
competent authorities to grant authorization for the GMO use in the EU.
Currently in the EU, 77 GM plants/products with a possible use in feed:
43 events of maize, 11 events of cotton, 15 events of soy bean, 4 events
of oilseed rape, a sugar beet, a potato and two micro-organisms have
temporary or full authorizations, granted under the GM Food and Feed
Regulation. Apart from the micro-organisms and the withdrawn potato,
these products are GM crops varieties of which most were produced to
exhibit resistance to certain herbicides, insect pests or both (first
generation GM products).
All of these GM varieties have been authorized for import and
processing. Two of the maize varieties have also been licensed for
cultivation, but only one is being grown commercially on a limited basis
in Europe. There are also currently GM plants developed with
significant intended alterations in agronomic properties (drought
resistance, salt tolerance etc), and in composition to enhance the
nutritional properties for health or growth benefits (second generation
GM products). A few of these products have been recently authorized to
be marketed in the EU: maize MON87460 modified for improving water
use efficiency; Soybean DP-305423 with modified seed fatty acid
content, specifically with high oleic acid and low linolenic acid contents,
and soybean MON 87769 which contains stearidonic acid (SDA; 18:4),
as an alternative source of an omega-3 fatty acid to help meet the needed
dietary intake of long-chain omega-3 fatty acids.
Recent efforts have focused on the development of second-generation
GM crops altered to increase output traits such as nutritional properties,
with the goal of improving human and animal nutrition and health.
Improving nutritional properties is realized by increasing the level of
desired substances, e.g., amino acids and fatty acids, or decreasing the
level of undesirable substances, e.g., antinutrients such as phytate (ILSI,
2007). In some cases, relatively complex genetic modifications have
been applied, resulting in substantial changes in plant metabolism and
composition.
A number of GM plants with characteristics intended to improve
nutritional benefits have been developed, Supplementary Material Table
S2 offer a list of nutritionally altered transgenic crops potentially
relevant to livestock.
These include GM plants in which:
a) a nutrient precursor, e.g., β-carotene,
(b) the concentration of specific nutrients, e.g., amino acids or fatty
acids,
(c) the digestibility of a specific nutrient, e.g., nitrogen or fiber,
(d) the content of a nutrient enhancer, such as enzymes (e.g., phytase)
(e) the content of substances with super-nutritional effects, e.g.,
prebiotics (inulin), has been increased,
(f) the concentration of an anti-nutritional factor, e.g., phytate
(g) the content of toxic substances, e.g., mycotoxins have been
decreased (EFSA, 2008).
Safety Assessment of Nutritionally Altered Feed:
Comparison of the GM plant with its isogenic counterpart is a key
component of safety and risk assessments and is seen as the most
reliable method to identify both intended and unintended effects of GM
plants. Once compositional equivalence has been established, livestock
feeding studies have not contributed to safety assessment (EFSA, 2008).
If compositional differences have been found upon comparison, a further
assessment of these differences for their safety and nutritional impacts
should be carried out, which may include an animal feeding study, if
applicable. For assessment of claimed nutritional benefits of second-
generation crops, livestock feeding studies may also be carried out and
should be conducted on a case-by-case basis. They can be conducted to
span or target the growing and/or finishing period until slaughter for
chickens, pigs and cattle, the major part of the lactation cycle for dairy
cows, and the laying cycle for layers.
Exposure assessment and post-market monitoring are important steps for
the risk assessment of GM crops, particularly those with novel output
traits that involve nutritional alterations (EFSA, 2013). Exposure
assessment is defined as ‘the quantitative estimation of the likely
exposure of humans and animals to the food and feed derived from GM
plants’ (EFSA, 2011a), and can help evaluate if compositional changes
have an adverse effect when food or feed is consumed. Post market
monitoring may be used to verify the validity of pre-market predictions
of consumption and use.
The International Life Sciences Institute (ILSI) also created a task force
that addressed the topic of safety and nutritional assessment of foods and
feeds that are nutritionally improved through biotechnology. A key point
addressed in the reports of this task force (ILSI, 2004, ILSI, 2008) is the
role of the comparative approach in safety assessment and how this can
be applied to nutritionally altered crops as they often differ significantly
in composition from non-GM crops. It was concluded that safety must
be addressed on a case-by-case basis and through the application of risk
analysis principles.
In addition, the ILSI task force gave detailed guidelines regarding study
design and considerations (e.g., use of appropriate controls) for
evaluation of crops with enhanced output traits fed to animals (ILSI,
2007). Reports by the European Food Safety Authority (EFSA) (EFSA,
2008, EFSA, 2011b, EFSA, 2013) and Flachowsky and Böhme (2005)
addressed safety assessment of GM plants fed to animals and can be
referred to for an overview of general principles for risk analysis and
safety assessment.
Animal Feeding Studies with Second-Generation GM Crops:
As outlined by the ILSI task force, livestock feedings studies should be
based on internationally established protocols and guidelines. Rapidly
growing animals have been suggested as a useful, sensitive model for
nutritional assessment of GM feed ingredients, monogastric animals
have been proposed for assessment of cereal grains (e.g., maize) and
protein sources (e.g., soybean meal), and growing or lactating ruminants
for assessment of forages. Table 4 includes recommendations from the
ILSI (2003) report” Best practices for the conduct of animal studies to
evaluate crops genetically modified for input traits” (GM plants of the
first generation). These recommendations can be applied to studies with
second-generation crops. However, other key considerations should be
taken into account and are detailed in the EFSA (2008) report on safety
and nutritional assessment of GM plants. These include experimental
design, use of an appropriate control, consideration of feed quality, and
use of an appropriate number of commercial varieties of feed in feeding
studies (EFSA, 2008).
The following sections address the topic of second-generation
nutritionally altered GM crops. We concentrate on crops altered to
increase amino acid and fatty acid levels and crops altered to address the
presence of antinutritional factors and increase bioavailability of
nutrients, specifically phosphorus. These three groups include the
majority of nutritionally altered crops from Table S2, relevant for animal
feed. Some other crops address nutrient characteristics relevant for
human food or industrial applications. Increase of vitamins and trace
elements is less important for animal feed because of the common
(over)supply of these micronutrients via the premix.
GM Crops with Enhanced Amino Acid Content:
Amino acids are the building blocks for animal protein. Most plants
have a poor balance of essential amino acids relative to the needs of
animals and humans. Cereals (maize, wheat, rice etc.) tend to be low in
lysine (Lys), whereas legumes (soybean, peas) are often deficient in the
sulphur rich amino acids, methionine (Met) and cysteine (Cys). As a
consequence of an imbalance in amino acid pattern in plant protein
sources for animal diets, essential amino acids, including Lys, Met,
threonine (Thr) and tryptophan (Trp) need to be supplemented in
synthetic form. The development of second-generation GM plants with
an increased content of limiting amino acid(s) provides an alternative to
the direct addition of synthetic amino acids in diets for monogastric
animals. To provide a nutritional assessment of a GM feed ingredient in
which the concentration of a specific nutrient such as an amino acid has
been increased, the following treatment structure is suggested (EFSA,
2008).
Comparison between T1 (negative control) and T2 (positive control) will
show the benefit of synthetic amino acid supplementation while the
comparison between T2 and T3 will demonstrate the efficacy of the GM
line. The comparison between T3 and T4 will provide further
comparisons between the use of a nutritionally enhanced GM line and
commercial varieties.
Such studies should be conducted in target species and diets would be
offered ad libitum while a range of animal performance endpoints need
to be measured. If the endpoints measurements comparing T2 and T3 are
similar, this indicates that the bioavailability of the nutrient enhanced in
the GM line is similar to that of the synthetic supplement and a
digestibility trial per se is not required.
However, when the endpoint measurements are markedly lower, this
could indicate a decreased bioavailability and/or the presence of an
unintended effect.
Lysine - Corn/Maize (Zea mays):
Genetically modified high Lys corn (HLC) (LY038 or LY038 x
MON810) has been developed by stable integration into the maize
genome of the cordapA coding sequence under the control of the maize
Glb1 promotor to direct the expression of the Corynobacterium
glutamicum-derived Lys feedback insensitive dihydrodipicolinate
synthase (cDHDPS) protein predominantly in the germ portion of maize
kernels. In a 42-day broiler study, the feeding values of grain from
LY038 or LY038 x MON810 were compared with a conventional
control (with similar genetic background) and another 5 conventional
maize hybrids.
The body weight gain, feed conversion ratio, carcass yields, and carcass
composition of broilers fed the high-Lys GM based diets were not
different from that of broilers fed the conventional maize L-Lys HCl
supplemented diet and better than the conventional based diets without
supplemental L-Lys HCl. There were no unexpected effects of GM
maize on health status or mortality.
In high-Lys high-oil corn (HLHOC), transgenic gene insertion leads to
the expression of a Lys-feedback-insensitive aspartokinase.
Aspartokinase catalyzes the first step in the Lys, Met and Thr
biosynthetic pathway through phosphorylation of aspartate.
Additionally, this genetic modification leads to the expression of a Lys-
feedback-insensitive dihydrodipicolinic acid synthase catalysing the first
reaction committed to Lys biosynthesis. Apparent ileal digestibility of
dry matter, energy, protein and amino acids was similar for HLHOC
compared with high-oil corn (HOC) in cannulated growing barrows.
There was no difference in apparent total tract dry matter and protein
digestibility in growing pigs fed HLC, HOC or common corn, but
apparent total tract digestibility of gross energy was lower for HLC
compared to conventional corn and HOC. Ileal AA digestibility was
numerically (not significant) lower in HOC versus HLC, with
intermediate results for HLC corn.
No effects of modified corn on growth performance and gain/feed ratio
were observed for diets with 65% HOC or HLHOC and for diets with
60% HLC or HOC in weaned piglets, nor for diets with 80% HLC or
HOC in growing-finishing pigs, all supplemented to the same level of
digestible essential amino acid content.
Amino Acids - Corn/Maize (Zea mays):
In genetically altered corn containing a glutamate dehydrogenase
(gdhA+) gene isolated from Escherichia coli, crude protein, leucine,
methionine, alanine, aspartic acid, glutamic acid and tyrosine were
increased. In female growing pig's apparent ileal amino acid
digestibility, total tract dry matter and nitrogen digestibility, and body
weight gain, were similar for the gdhA+ transgenic corn and non-
transgenic corn supplemented with pure amino acids.

Methionine - Lupin (Lupinus angustifolius L):

By raising the sulphur amino acid content of lupin, the nutritive value of
lupin for animal feeds is increased. In high-Met lupin of the Warrah
variety, met content was doubled through the introduction of a chimeric
gene encoding the sulphur-containing amino acid-rich sunflower seed
albumin. Apparent ileal digestibility of individual amino acids in 35–40
days old female broilers was similar for conventional and transgenic
lupins, although ileal digestibility of Met tended to be higher in
transgenic lupins. In female broilers from 6 to 20 days of age, weight
gain and feed intake were not different for a maize-soybean meal diet
with 25% transgenic lupins or conventional lupins with supplemented
methionine.
A doubling of the Met content of the Warrah variety of the narrow-
leaved lupin has been achieved by insertion, facilitated by the use of a
DNA transfer vector from the plant tumor-inducing Agrobacterium
tumefaciens, and expression of a sunflower protein coding gene within
the lupin genome. In juvenile red seabream (Pagrus auratus) feed intake
and body weight gain were similar when feeding a transgenic Warrah, a
non-transgenic Warrah and a non-transgenic DL-Met supplemented
Warrah whole seed meal diet with adequate or low protein contents.
Protein digestibility of non-transgenic and transgenic lupin kernel meal
was not different.
In lupin cv Warrah, Met content was almost doubled and cysteine
content slightly decreased through the introduction of a chimeric gene
specifying seed-specific expression of a sulphur-rich sunflower seed
albumin.
In sheep fed a cereal hay-based diet with 35% unmodified or transgenic
lupin seed, there were no differences in organic matter digestibility,
rumen microbial protein synthesis and in sacco degradability of dry
matter. In sheep fed the transgenic lupin seed, the rate of wool growth,
the sulphur concentration of wool, and live weight gain were higher and
plasma cysteine and Met concentration tended to be higher while plasma
urea N was lower, compared to the unmodified and supplemented lupin
seed.
Tryptophan - Rice (Oryza sativa):
In high Trp-rice, a transgene (OASAID) encoding a feedback-insensitive
alfa subunit of rice anthranilate synthase was expressed resulting in the
accumulation of Trp in calli (mass of unorganized parenchyma cells)
and leaves and a 50% higher content of Trp.
In male broilers fed low protein diets based on rice and soybean meal for
21 days, feed intake and body weight gain were similar for the non-
transgenic Trp supplemented rice diet and the high transgenic Trp-rice
diet and significantly higher compared with the non-transgenic non-
supplemented rice diet.
Tryptophan - Soybean (Glycine max L.):
In high Trp-soybean seeds, the gene encoding a mutated feedback-
resistant alfa subunit of rice AS (OASAID) under the control of either a
soybean glycinin gene promoter or the 35S promoter of cauliflower
mosaic virus for seed-specific or constitutive expression, respectively,
was introduced by particle bombardment.
Rainbow trout (Oncorhynchus mykiss) were fed diets with 40% defatted
seed meal for 6 weeks from 11 weeks after hatching (initial body weight
of 0.7 g). Body weight was not different for the non-transgenic seed
supplemented with Trp and the transgenic seed meal and significantly
higher compared with the non-transgenic non-supplemented seed diet.
Conclusions on GM Crops with Enhanced Amino Acid Content:
From the studies done so far, it can be concluded that the bio-efficiency
of diets with GM plants with increased amino acid content does not
differ from that of conventional plants supplemented with synthetic
amino acids. However, several do not meet the general design with
positive and negative control treatments as proposed by EFSA (2008)
and hence results are inconclusive. For GM plants with modified amino
acid content, specification of the complete amino acid pattern and level,
and the (ileal) digestibility is required for safe inclusion in animal diets.
Young rapidly growing broilers and pigs would be the most sensitive
species for a deficient amino acid supply.
GM Crops with Modified Fatty Acid Content:
Fat is an important energy source for humans and animals. Fats consists
of a complex mixture of triacylglycerol molecules that can differ greatly
from one another in their chemical and physical properties and have a
wide range of health benefits. In oilseed plants, the composition of the
fatty acids of lipids has been modified mainly for industrial purposes,
rather than for their feeding value in animal diets.
Modification of fatty acids is also performed to deliver healthier
products to human consumers, for instance low- and zero-saturated fat
oils and oil containing stearidonic acid (SDA), a polyunsaturated fatty
acid (PUFA).
To provide a nutritional assessment of a GM feed ingredient in which
the concentration of fatty acids has been changed, a similar treatment
structure as for testing amino acid enriched GM feed ingredients has
been suggested (EFSA, 2008).
Soybean (Glycine max L.):
Soybean DP-305423-1 was modified by the insertion of the gm-fad2-1
gene fragment, resulting in greater concentrations of oleic acid by
suppression of FAD-2gene expression. The FAD-2 gene encodes an n-6
fatty acid desaturase enzyme that catalyzes desaturation of oleic acid
(18:1) to lineoleic acid (18:2), resulting in increased levels of oleic acid
and decreased levels of linoleic acid, linolenic acid and palmitic acid.
Oil produced from DP-305423-1 soybean has a better oxidative stability
which contributes to improved frying performance. Male and female
broilers were fed diets containing processed fractions of DP-305423-1
transgenic soybeans, non-transgenic soybeans with a comparable genetic
background and three other commercial non transgenic soybeans for 42
days. No differences in body weight gain, feed efficiency, mortality,
organ (liver and kidney) and carcass (breast, thigh, leg, wing and
abdominal fat) yield were observed.
Laying hens were used to evaluate the nutritional equivalence of
soybean meal derived from DP-305423-1 transgenic soybeans. Hens fed
diets with DP-305423-1 transgenic soybean meal had similar body
weight, hen-daily egg production, egg mass, feed consumption and feed
efficiency in comparison to hens fed the non-transgenic near-isoline
soybean meal and non-transgenic commercial soybean meals. In
addition, egg (component) weights were not different for the four
soybean meal diets.
SDA-soybean oil originates from genetically modified soybeans
producing relatively high concentrations of stearidonic acid (SDA;
C18:4n-3). In broilers fed a diet with SDA-soybean oil, near-isogenic
conventional soybean oil or fish oil (4.5% in grower diet and 5% in
finisher diet), there were no differences in feed intake, weight gain and
feed efficiency between the diets. Carcass and total breast weights
(including fat content) were not different for the SDA and conventional
soybean oil diet. The SDA, EPA (C20:5n-3; eicosapentaenoic acid),
DPA (docosapentaenoic acid; C22:5n-3), DHA (docosahexaenoic acid;
C22:6n-3) and total n-3 concentration in meat were higher in SDA than
in the conventional soybean oil diet.
Also in broilers fed a diet with 5% SDA-soybean oil from 28 to 42 days
of age, there were no differences in feed intake, weight gain and feed
efficiency but an increase in total n-3 content (predominant DPA) in
breast, tender and thigh compared to broilers fed a diet with
conventional soybean oil.
In transgenic SDA-enriched soybean, genes for Δ6 desaturase enzyme
(from the commercially grown fungus Mortiella alpine) and for Δ15
desaturase enzyme (from canola (Brassica napus)) were inserted to
accumulate stearidonic acid (Ursin, 2003).
In multiparous rumen-fistulated Holstein cows, abomasal and ruminal
infusion of SDA-soybean oil were compared with control (no infusion)
in a 3x3 Latin Square design. Abomasal and ruminal infusion of SDA-
soybean oil did not affect milk production, milk protein percentage and
yield, lactosepercentage and yield and milk fat yield. After ruminal
infusion of SDA-soybean oil there was almost no transfer of SDA to
milk fat. However, after abomasal infusion of SDA, 39% of SDA was
transferred to milk fat and n-3 fatty acids concentration was found to be
more than 5-fold greater. Hence, SDA-soybean oil in cow diets, when
properly protected against ruminal biohydrogenation, may enhance
omega-3 fatty acid concentration in milk.
Rapeseed/Canola (Brassica napus):
In the transgenic full-fat rapeseed cultivar line TM5, insertion of the
acyl-thioesterase gene of Cuphea lanceolata (Cigar flower from Central
America) altered fatty acid metabolism and resulted in a higher content
of myristic (C14:0) and palmitic (C16:0) acid and lower content of oleic
(C18:1) acid in the rapeseed plant. This construct was developed for
industrial purposes (mineral oil replacement), the coproducts such as
press cake and extracted meal are intended to be used as feedstuffs. In
growing-finishing pigs, fecal digestibility was not different for diets with
15% TM5 full-fat rapeseed or conventional full-fat rapeseed, but there
was a tendency to a lower feed intake and weight gain for the TM5 full-
fat rapeseed diet, presumably due to the 50% higher glucosinolate
content of the TM5 rapeseed. In back fat and intramuscular fat, myristic
acid concentration was higher and oleic acid content was lower in the
meat of pigs fed the TM5 full-fat rapeseed diet.
In conclusion the genetic engineering of plants with the aim to change
their composition might be associated with an increase of undesirable
substances. Glucosinolates are toxic compounds which cause a bitter
taste to livestock diets, whilst indirectly they can also account for off-
flavors to animal-derived food products such as eggs after uptake by the
animal from digested feed and transfer to such products.
This underscores the importance of including such undesirable
components into the compositional analysis of new canola varieties as
recommended by the OECD Task Force's Consensus document on key
nutrients and anti-nutrients for such analyses.
Conclusions on GM Crops with Modified Fatty Acid Content:
These studies indicate that in general genetic modification of the fatty
acid content of plants and their use as feed ingredients did not influence
the production performance, i.e. production of meat, eggs, and milk.
However, changes in fatty acid patters of plants were reflected in the
fatty acid composition of the animal products. The consequences of
human consumption of these animal products should be included in the
evaluation of these GM crops.
GM crops with a Reduced Content of Antinutrients:
Antinutrients are endogenously produced plant substances that inhibit
the utilization of nutrients and negatively impact animal health and
production (for common plant antinutrients see Table 5. Monogastric
animals (e.g. poultry and pigs) are generally more affected by
antinutrients than ruminants as they lack the protective effect of the
rumen microflora which binds and degrades antinutrients (ILSI, 2007).
This is problematic as most unprocessed feeds of plant origin, e.g. maize
and soybean, contain antinutrient levels that limit the performance of
sensitive animal species (Cowieson, 2005, ILSI, 2007).

Nonetheless, antinutrients like tannins may have beneficial effects, e.g.

by providing protection against pests during production and storage of
feed and by improving nitrogen digestion in ruminants as well as
decreasing parasite loads (ILSI, 2007, Thompson, 1993).The main
strategies for alteration of antinutrient levels in animal feed include the
use of physical or enzymatic means to reduce antinutrient levels (e.g.,
treatment of seeds to reduce phytate levels) and supplementation of
nutrients (e.g., protein for protein inhibitors, vitamins, for vitamin
antagonists, iron for gossypol, and phosphorus for phytate).
These approaches however are costly and may limit extensive
commercial use. Recently, more emphasis has been put towards the use
of transgenic approaches as an alternative to address antinutrients in feed
(ILSI, 2007). Whereas such products are primarily intended to be used
as livestock feedstuffs, their safety for both human and animal
consumers will be jointly evaluated during the pre-market regulatory
safety assessments, such as in the European Union and USA.
Phosphorus, Phytate, and Phytase:
Phosphorus (P) is an important mineral that has many roles within the
body. A significant proportion of phosphorus in plant products,
especially cereal grains and oilseed meals (60–80%), is stored as phytate
(also known as inositol hexakisphosphate or phytic acid). Because
monogastric animals such as pigs, poultry, and fish lack the enzyme
phytase, phytate bound phosphorus is poorly utilized by these species. It
passes through the digestive tract largely undigested and ends up being
excreted in the manure. The undigested phosphorus can then accumulate
in the environment.

Additionally, phytate also binds amino acids, proteins, and minerals

(e.g., calcium, iron, magnesium, manganese, and zinc), preventing their
utilization in monogastric animals (ILSI, 2007).
The main approach to reduce the effect of phytate involves dietary
supplementation with phytase. which consistently resulted in improved
growth performance, P utilization and bone mineral concentration in
pigs and poultry.
Other methods include pre-treatment of grains to activate endogenous
phytase, mutation of enzymes involved in phytate biosynthesis to reduce
seed phytate levels, breeding of low phytate crops, and transgenic
alteration to produce low phytate crops or crops expressing a phytase
gene.

The last approach is gaining ground as it might be a successful, cost-

effective way to improve the bioavailability of phosphorus in animal
feed. This method involves expression of the phytase gene (phy) of
bacterial or fungal origin in plants., Fungi have been more widely used
as they are stable at a wide range of temperatures and pH. The fungal
phyA genes from Aspergillus niger and Asperigillus fumigatus are
common examples and have been applied to the development of
transgenic alfalfa, canola, maize, rice, soybean, tobacco, and wheat
crops.
The efficacy of this approach has first been demonstrated by using
tobacco seed with engineered phytase gene from Aspergillus niger to
enhance P release and growth performance in chickens.

Several different genetic engineering methods have been used to

produce recombinant plants expressing the phy gene including fusing the
phy gene to the plant endoplasmic reticulum target sequence,
introduction of the phy gene by microprojectile bombardment of cell
suspension culture and placing the phy gene under control of different
promoter sequences. For modification of phy gene expression once it has
been introduced into the plant, strategies include modifying the codon of
the gene according to the usage of the host plant and directing soluble
proteins to a specific location within the plant.

Transgenic Phytase Crops:

It has been well-established that the inclusion level of dietary phytase to
the diets of pigs and poultry can influence the results among and
between species. Therefore, careful consideration needs to be given to
this aspect when providing diets with supplemented phytase or
transgenic phytase crops.
Results from studies so far have demonstrated that feeding phytase
transgenic plants may be as effective as supplementation of microbial
phytase to improve utilization of phosphorus as well as a number of
other nutrients in monogastric animals. Nonetheless, several studies lack
adequate (negative) control treatments to unequivocally proof that
phosphorous was limiting in control diets without added phytase or
inorganic phosphorous.
In addition, to allow safe and effective inclusion in practical diets,
content and efficacy of phytase in transgenic maize should be quantified
and guaranteed to assure an adequate inclusion level of transgenic maize
to meet the phosphorous requirements. Inadequate phytase content and
phosphorus availability would imply a nutritional risk of transgenic
plants.
This could be due to a too low inclusion level of the GM feed ingredient,
lower phytase content than expected or loss of phytase because of heat
treatment during feed processing. Presumably, young rapidly growing
broilers and pigs, and laying hens would be the most sensitive species
and skeleton disorders and locomotion problems the most likely signs, in
addition to a general loss in appetite and production performance.
Currently, no transgenic phytase crops are approved on the European
market for either growing or marketing. According to the International
Service for the Acquisition of Agri-Biotech Applications (ISAAA)
database, six transgenic phytase crops are presently approved: five
canola crops for food and feed in the United States and one maize crop
in China. Additional transgenic phytase crops are currently not on the
market and have only been used for research purposes.
The safety of transgenic phytase canola has been established for
approved crops. Several studies investigated the inclusion of transgenic
phytase maize in diets for broilers, laying hens, roosters and pigs and
observed no adverse effects associated with transgenic phytase maize.
The Final Word Upon GM Feed:
Second-generation crops offer a potential alternative to addressing
nutritional imbalances in animal feed.
The EFSA and ILSI provide guidelines regarding the safety and risk
assessment of feed altered through biotechnology as well as best
practices for conducting studies to evaluate second-generation GM
crops. Additional, standardized pre-market (registration) studies based
on these guidelines are needed to further elucidate the safety as well as
potential of second-generation transgenic crops to sustain and improve
the health and nutrition of livestock. Studies should include relevant
positive and negative control treatments to assure the response of the
animals to the specific nutrients.
In addition, studies should provide quantification of the content and
bioavailability of target nutrients in the GM products for their scientific
based and cost-effective inclusion in animal diets.
The potential contribution of increased phytase levels in GM crops to
improve the phosphorus supply has been demonstrated. No nutritional
imbalances are expected provided that their quantitative contribution to
the phosphorus supply of the target animals is adequately established.
PMM studies to monitor the risk of phosphorus deficiencies can best be
conducted in fast growing young broilers and pigs, with emphasis on
anorexia (reduced feed intake and growth) and locomotion problems as
primary clinical signs. Plasma inorganic phosphorus can be additionally
determined.
However, these signs are not specific for GM crops but would be general
indicators of potential phosphorus deficiency, and can only be
interpreted in relation to diet composition. Therefore, specific case-
control studies would be recommended.
The potential contribution of several GM modified crops with increased
levels of specific amino acids to the amino acid supply of pigs and
poultry has been demonstrated. No nutritional imbalances are expected
provided that the quantitative contribution of these GM crops to the
digestible amino acid supply of the target animals is adequately
established. The risk of amino acid deficiencies can best be monitored in
fast growing young broilers and pigs, using reduced growth rate and lean
meat content as (clinical) signs. Other specific health problems related to
mild or moderate amino acid deficiencies are not too be expected.
The fatty acid composition of oil seed plants is genetically modified for
industrial purposes or to deliver healthier products to human consumers.
In general, most of the oil is extracted and the protein rich meal is
included in animal diets. Hence, the changes in fatty acid composition
may not be reflected in feed composition and have little effect in the
animals unless full fat seeds and beans, or extracted oil is included in the
diet. The variation in fatty acid pattern in animal diets due to use of fatty
acid altered GM crops presumably is much less than the variation in the
inclusion of different amounts of soya bean, rapeseed, and palm
products, being the major fat sources in animal diets. Consequently,
PMM of consequences of GM changes in fatty acids presumably would
be overruled by other sources of variation.
It is important to monitor potentially negative concomitant changes in
the composition of plants as a result of desired GM modifications. This
aspect is not specific for nutritionally altered second generation crops.
Any GM modification is mediated by changes in plant proteins and may
potentially have negative side effects. These aspects are addressed
elsewhere in this issue.
The recently concluded GMSAFOOD project proposed a novel strategy
in which experimental data undergo machine learning clustering and
neural network analyses to identify potential biomarkers capable of
detecting unforeseen health risks related to GMOs. Data from extended
and transgenerational feeding studies with rats, mice, fish and pigs of
different ages and food chain trial data on rats were entered into a newly
established database containing raw data. Although adverse health
effects related to GMO exposure were not identified in those
experiments, the applied machine learning methodology would enable
the identification of the GMO exposure related potential biomarkers.
All in all, the main difficulties to find out any potential health effect is
related to the availability to have multiple sources of data, to capture a
wide range of possible effects and to have a general approach or system
that could be employed for performing case-specific monitoring.
Actually, there is not a single tool and guidance enabling harmonized
monitoring/surveillance systems for monitoring animal health, that could
be also used to collect data in the context of GMO fed animals. On the
other hand, it is considered out of proportion to set up a systematic
process of data collection only for GM issues. Post Market monitoring
context could be a useful tool to gather evidences, but it is realized only
for specific cases where the premarket safety assessment highlighted a
residual uncertainty.
Concerning allergenicity, there are no case reports of allergic reaction or
immunotoxin effects resulting from feeding currently EU approved GM
feed products to livestock as compared with corresponding non-GM
feed.
The (assumed) low proportion of affected livestock animals in the field,
the lack of diagnostic means and the unspecific clinical signs make it
difficult to identify disease signs as a consequence of immunotoxicity
and allergenicity of GM proteins. Very little research focused on
determining immune parameters of livestock animals in GM feeding
trials, and especially parameters involved in allergic reactions.
Consequently, no information is available regarding both the number of
case reports in farm animals and the underlying mechanisms explaining
the physiopathology of the adverse effects observed. The development
of validated and standardized (laboratory) animal models for the
assessment of allergenicity in farm animals is an open issue. A strategy
of monitoring could focus on case-control studies in regard to specific
age groups (i.e. weaning, change of group composition, change of diets)
or extremes of production systems in different species (i.e. high
production pig systems, veal calves, etc.). Especially for second
generation of GM plants, where novel proteins are introduced or where
the composition is changed, e.g. in which the content of proteins is
increased, post-market monitoring could be applied if the pre market risk
assessment showed residual uncertainty related to allergenicity. When
technically possible, a PMM could involve screening of animals for IgE-
sensitization to the protein by in vitro tests or by skin prick testing. Since
specific IgE levels in blood are not always related to allergy, careful
evaluation of clinical findings (in first instance presence of diarrhea,
reduced growth performance, weight loss) and exclusion of differential
diagnoses should take place.
Regarding horizontal transfer, the risk of gene transfer from GM crops
to microbes of mammalian cells must be weighed against the relative
risk of a similar event occurring in nature. Transfer of GM traits to
microorganisms by transformation is a limited possibility and is only
significant if the new trait is expressed and confers selective advantage.
There are no case reports of horizontal transfer of an intact and
functional gene to mammalian cells from GMP currently approved in
EU. In fact, according to this report, a precise prediction of the fate of
dietary DNA in mammalian cells is currently unachievable in the
majority of studies. For this reason, such kind of findings should not
form the basis of risk assessment of horizontal DNA transfer derived
from novel GM food and feed. It is also reasonable to suggest that the
risk of transfer of transgenic DNA is equal to that for non-transgenic
DNA and that unintended horizontal gene transfer is unlikely to raise
health concerns.
Mycotoxins are unavoidable contaminants, which cannot be completely
eliminated from the agri-food chain, that have economic and health
impact. Examining in depth the available scientific literature, it is
recognized that GM crops, and their use in the feed chain, confer a
reduction of fumonisins contamination and this can be considered as an
advantageous side effect with positive consequences in GM-crop-
derived feed safety. Other Fusarium toxins (e.g. deoxynivalenol and
zearaleone) and aflatoxins are not systematically controlled and more
field studies should be performed also to verify if these reduction
imbalances the competition among different mycotoxins producing
fungi. As mycotoxin contamination reduction is only a positive side
effect of Bt GM crops, no mandatory PMM can be asked for this topic.
However, these data could have an impact on the assurance of safety and
quality and promote a greater insight into the occurrence of health and
welfare impacts of GMOs.
A better understanding of the effectiveness of the GM feeding positive
effect as regards mycotoxin contamination should be attained by a pan
European collection of mycotoxin occurrence, animal exposure to insect
resistant GM crops and animal performance data. Feasible monitoring
studies should monitor parameters easily available to stakeholders.
For this purpose, the monitoring of aflatoxin B1 content in GM maize
derived feed (which are data available from EU official control), and
aflatoxin M1 in milk from GM maize-fed dairy cattle (which is also
available from EU official controls) should be put in correlation
respectively with growth rate and dairy cattle milk production to
ascertain any kind of correlation.
As for the second-generation nutritionally altered crops, the complex
genetic modifications applied to create second-generation crops lead to
substantial compositional changes, that require special consideration
with regard to safety and nutritional assessment. The potential
contribution of increased phytase levels in GM crops to improve the
phosphorus supply does not envisage nutritional imbalances provided
that their quantitative contribution to the phosphorus supply of the target
animals is adequately established. The risk of phosphorus deficiencies
can best be monitored in fast growing young broilers and pigs, and
laying hens, with emphasis on locomotion problems. However, these
signs are not specific for GM crops but would be general indicators of
potential phosphorus deficiency and can only be interpreted in relation
to diet composition. The potential contribution of several GM modified
crops with increased levels of specific amino acids to the amino acid
supply of pigs and poultry not envisage nutritional imbalances provided
that their quantitative contribution to the digestible amino acid supply of
the target animals is adequately established. The risk of amino acid
deficiencies can best be monitored in fast growing young broilers and
pigs, but the general loss in growth performance would be difficult to
relate to the use of GM ingredients. The variation in fatty acid pattern in
animal diets due to use of fatty acid altered GM crops presumably is
much less than the variation in the inclusion of different amounts of
soybean, rapeseed, and palm products, being the major fat sources in
animal diets.
Consequently, post market monitoring of consequences of GM changes
in fatty acids would be overruled by other sources of variation.
Networks of stakeholders such as professional and farmer’ organizations
might serve as primary contact point for dissemination and validation of
pertinent information on possible animal health impacts. Furthermore, to
give support to post market monitoring of second-generation GM crops,
when required based on the safety assessment, it would be useful to
organize a EU-wide large scale data collection of critical key health
indicators (positive and negative) as a baseline for monitoring
potentially negative changes as a result of the introduction of second
generation GM derived feed.
Comparable observations are also reported by ADAS (2015), whose
report provides a systematic review of existing monitoring program and
an inventory of data collection sources that may be useful for PMM of
GM food and feed.
In theory, an approach based on monitoring and information exchange
would greatly benefit from the linkage between health data with
consumption figures, because this would advance our comprehension of
the possible relationship between feed consumption by livestock and
associated impacts on the health and welfare of animals, not only for
GM feed, but for feeds in more general terms.
Post Market Monitoring (PMM) of Food and Feed Derived from
GM Plants:
GMOs are authorized only if considered safe for human and animal
health and the environment. Nevertheless, their routine surveillance has
been considered necessary as precaution in those cases when there are
residual uncertainties deriving from the pre-market safety assessment
and to detect unforeseen effects.
Towards this end, all applications for releasing them into the
environment or marketing GMOs as food, feed and derived products,
must propose a PMM for each specific GMO for which a marketing
application has been filed. This plan is part of the EU authorization
decision and the applicant must implement it and regularly report on it to
the EU authorities. The objective to be achieved and the general
principles to be followed to design the monitoring plan are described in
the annex VII of the Directive 2001/18/EC: “The objective of a
monitoring plan is to: i)confirm that any assumption regarding the
occurrence and impact of potential adverse effects of the GMO or its use
in the environmental risk assessment (e.r.a.) are correct, and ii) identify
the occurrence of adverse effects of the GMO or its use on human health
or the environment which were not anticipated in the e.r.a.” The first
type of monitoring activities is also referred to as “case-specific” given
that it focuses on a specifically identified issue identified during pre-
market risk assessment (e.g. the development of pest insect resistance to
an insect-resistant crop) whilst the second type is referred to as “general
surveillance”.
Applications concerning food/feed uses and import and processing do
not require scientific information on possible environmental effects
associated with the cultivation of the plant given that this outside the
scope of the application. The program of the environmental PMM will
depend on the level of the release and exposure on the environment. In
fact, the EFSA guidelines differentiate between general surveillance
plans when GM is for cultivation and when applications are intended for
import/processing.
In the latter case, general surveillance plans need to consider the
modified characteristics specific to the GM plants in question, their
intended use and the receiving environment. In the case of non-viable
GM material (e.g. derived products not containing any living GMOs)
and according to Directive 2001/18/EC, the applicant does not have to
provide any environmental monitoring plan (including general
surveillance). In the case of imported GM products containing viable
propagating material, general surveillance plans should consider that,
appropriate management systems to restrict environmental exposure if
substantial loss, spillage from the establishment is possible.
Case-specific Post Market Monitoring should be required for food and
feed derived from GM plants only in specific cases, such as for foods
with altered nutritional composition and modified nutritional value
and/or with specific health claims. In the particular case of various
vegetable oils with modified fatty acid composition, the PMM thus
requested had to complement the thorough pre-market risk assessment
with further information on the intake of the product by consumers.
Given that pre-market risk assessment studies have some attendant
uncertainties regarding coverage of the breadth of prospective
consumers’ health status and intake levels, a PMM should therefore
address the following general questions:
(i) Is the product’s use as predicted/recommended?
ii) Are the known effects and side effects of the product as predicted?
iii) Does the product induce unexpected side effects?
The design of a PMM plan for food and feed should enable the
collection of trustworthy data that can be used to verify the validity of
pre-market predictions of consumption and use, and to link any potential
health impacts to the consumption of the particular GMO food or feed,
with detailed reporting's (i.e. crop, GM trait, changes in the
consumptions, etc.), about any (adverse) effect on human and animal
health and animal performances.
Although PMM may be required for Novel Foods and Feed in general, it
may be difficult to perform in the case of a GM commodity crop like
those currently in the marketplace. Therefore such a program appears
feasible only when the composition and the nutritional value is
substantially changed as compared to the non-GM conventional
counterpart and sold separately through identity-preserved chains, for
example.
 Case Studies
Now that we have covering the basics about genetically modified
organisms let us delve into the further details that they individually
possess.

Golden Rice:
All plant tissues that accumulate high levels of carotenoids have
mechanisms for carotenoid sequestration, including crystallization, oil
deposition, membrane proliferation or protein-lipid sequestration. It has
been shown that lipid accumulation can be a driver of carotenoid
formation by acting as a lipophilic sink.
The non-carotenogenic starchy rice endosperm, on the other hand, is
very low in lipid and apparently lacks any such means for carotenoid
deposition. It was also doubtful whether Golden Rice would have the
necessary precursors for carotene biosynthesis present and available in
the grains, with many believing that the whole, multi-step carotenoid
biosynthetic pathway was completely absent from the endosperm.
This explains why a long research phase preceded the achievement of
the proof-of-concept for Golden Rice. By the early 1990s, the data
accumulated became encouraging enough for Profs Peter Beyer and Ingo
Potrykus to gather forces and dare to tackle this feat. Their breakthrough
showed that only two transgenes were required to turn Golden Rice into
a reality.
The first transgene encodes a plant phytoene synthase (PSY), which
utilizes the endogenously synthesized geranylgeranyl-diphosphate
(GGPP) to form phytoene, a colorless carotene with a triene
chromophore. The second gene encodes a bacterial carotene desaturase
(CRTI) that introduces conjugation by adding four double bonds.
Between 1993 and 1999, collaborative research between Peter Beyer and
Prof Peter Bramley (Royal Holloway College, UK), was funded through
EU networks B102-CT-930400, B104-CT97-2077 and FAIR CT96
1633.
Working with genetically modified tomatoes, Peter Bramley established
the advantage of using a single phytoene desaturase gene (bacterial
CRTI), rather than introducing multiple plant desaturases. The combined
activity of PSY and CRTI leads to the formation of lycopene, which is a
red compound, its color stemming from its undecaene chromophore, as
is well established in tomato fruit.
However, lycopene has never been observed in any rice transformant
and different genetic backgrounds. Instead, α- and β-carotene are found
together with variable amounts of oxygenated carotenoids
(xanthophylls), such as lutein and zeaxanthin.
The carotenoid pattern observed in the grain's endosperm revealed that
the pathway proceeded beyond the end point expected from the
enzymatic action of the two transgenes alone. The findings are explained
in some detail below.
 Figure explanation: Filling a biosynthetic gap: Pathway elements in
green are functional in wild-type rice grains. Thus, the GGPP precursor
molecule is being synthesized and lycopene can be cyclized. Elements in
blue, including the blue box, are effectively absent. Introduction of the
enzyme's phytoene-synthase and the bacterial desaturase CRTI fills the
biosynthetic gap created by the absence of the blue elements.

The explanation is that enzymes further down the pathway, such as

lycopene cyclases (LCYs) and α- and β-carotene hydroxylases (HYDs),
are still being produced in wild-type rice endosperm, while PSY and one
or both of the plant carotene desaturases —phytoene desaturase (PDS)
and ζ-carotene desaturase (ZDS)— as well as the cis-trans isomerases,
namely ζ-carotene cis-trans Isomerase and carotene cis-trans isomerase
are not. Synthesis of lycopene by PSY and CRTI in transgenic plants
provides the substrate for these downstream enzymes and consequently
enables the formation of the observed products.
The fact that a PSY transgene alone is sufficient for phytoene
accumulation but does not lead to desaturated products is evidence for
the absence of at least one active desaturase, namely PDS. Similarly, the
expression of CRTI alone did not result in any colored compounds in the
rice endosperm, because of the lack of PSY activity. As stated above, the
advantage of the CRTI desaturase lies in the fact that it can perform the
entire reaction sequence from phytoene to lycopene on its own, while
plants employ two desaturases and two cis-trans isomerases to achieve
the same outcome. This reduces the number of transgenes required to
only two.
The need for CRTI apparently conflicts with the presence of PDS and
ZDS transcripts in wild-type endosperm, as shown by quantitative RT-
PCR analyses. This could be due to low level presence of the enzyme
rather than its mRNA. Due to the low-level expression, the complex
reaction mechanisms of PDS and ZDS, and the unavailability of
radioactive carotene substrates, the investigations were done using a
transgenic approach rather than in vitro reactions. The tissue-specific
expression of the PDS/ZDS system, instead of CRTI, in rice endosperm
resulted in the formation of colored carotenoids, showing that the rice
endosperm provides the complex requirements for the activity of the
plant desaturases.
The primary sequence of CRTI is unrelated to the plant-type desaturases.
Its structure has been partially resolved and the reaction mechanism
investigated. Clearly, CRTI is simpler than plant-type desaturases. CRTI
employs molecular oxygen directly as an electron acceptor, while the
plant enzyme utilizes plastoquinone for this purpose, and is thus linked
to and dependent on complex redox chains. This electron path is also
indirectly linked to molecular oxygen as the terminal electron acceptor
via an oxidase identified through the immutans mutation of Arabidopsis.
This redox pathway is especially important in non-green carotenoid-
bearing tissues, while the photosynthetic electron transport is thought to
play an analogous role in chloroplasts. Moreover, CRTI does not form
poly-cis-configured intermediates, as plant desaturases do and therefore,
cis-trans isomerases are not required. CRTI is also capable of
introducing all four double bonds in one step.
Clearly, lycopene cyclase activity relies on the expression of the
respective rice genes in the endosperm, just as the occasional formation
of xanthophylls, catalyzed by the divergent class of β-carotene
hydroxylases. In all rice genetic backgrounds tested so far,
complementation with these activities is not required to proceed down
the pathway. Moreover, the activity of rice LCYs is obviously not rate
limiting, since lycopene does not accumulate. Thus, Golden Rice is
yellow because of the activity of intrinsic rice cyclases.

Golden rice.
First generation:
The first breakthrough in the development of Golden Rice was the result
of a collaboration between Peter Beyer and Ingo Potrykus, and was
obtained around Easter 1999. This paper provided the proof that β-
carotene could be produced in the rice grain. At the time it became
evident already that only phytoene synthase and carotene desaturase
(CRTI) were needed to get the pathway going, while lycopene cyclase
was not required.
These initial experiments were carried out with a Japonica (round grain)
rice cultivar. Later, this was also achieved in Indica (long grain)
cultivars, a finding that has been confirmed many times over the years,
by crossing the trait into a number of varieties by breeding.
With the proof of concept in hands, the scientists immediately proceeded
to develop ways to improve the production and accumulation of
carotenoids in the seed, as it was recognized that at the levels attainable
at the time (1.6 μg/g) Golden Rice would not be able to cover the daily
provitamin A requirements of the target population in the absence of a
more varied diet.
While some population strata in SE Asia do consume more varied diets,
many of the poorest do not, and in fact, in some rural populations rice
makes up more than 80% of their daily caloric intake.
These efforts led to the development of what we could call the first
generation of Golden Rice (after the proof of concept), also known as
GR1. This version only contained the phytoene synthase gene from
daffodil and the carotene desaturase gene from the bacterium Pantoea
ananatis (previously known as Erwinia uredovora). Further, in this early
version both transgenes were expressed only in the rice endosperm (by
placing the genes under the control of the endosperm-specific gt1
promoter). The levels of carotenoids obtained in the field amounted to
an average of 6 μg/g (about 4 times higher than the prototype), probably
due the availability of large numbers of transformation events to select
from and the use of the tissue-specific gt1 promoter to drive CRTI
expression, while the constitutive 35S promoter had been used in the
proof-of-concept prototype.
New generation:
The first generation of Golden Rice showed that it was possible to
produce provitamin A in rice grains, but it was recognized that to
combat vitamin A deficiency higher β-carotene levels would be
required. As only two biosynthetic transgenes are required in the
process, the logical approach was to identify the bottleneck of the
biosynthetic pathway and fine-tune the enzymatic activities of the two
gene products involved, phytoene-synthase (PSY) and carotene-
desaturase (CRTI).

In multi-step biosynthetic pathways there is generally a rate-limiting step

that controls the flux through the whole pathway. This can be overcome
by either increasing the amount of the rate-limiting enzyme or by using
one that is more active. It was established that in this case was PSY and
not CRTI. Experimentation with PSY genes from different sources
identified the maize and rice genes as the most efficient in rice grains, a
result that has was confirmed later at the enzyme level.
This led to the second generation of Golden Rice lines, often referred to
as GR2, capable of accumulating up to 37 μg/g carotenoids, of which 31
μg/g was β-carotene, as compared to the first generation, where only 1.6
βg/g were obtained.
Given that bioconversion of β-carotene from Golden Rice is a very
efficient process, as highlighted on the homepage of this website, a
typical diet containing GR2 has a great potential to help alleviate
vitamin A deficiency-induced diseases.
Below is the progress of golden rice development. The image clearly
shows the progress made since the proof-of-concept stage of Golden
Rice. The new generation, also known as GR2 contains β-carotene levels
that will provide adequate amounts of provitamin A in children's diets in
SE Asia.
Flavr Savr Tomato:
The FLAVR SAVR tomato was the first genetically engineered crop
product to be commercialized. The research and marketing efforts that
produced the FLAVR SAVR tomato resulted in scientific success, a
temporary sales success, and then commercial demise. The FLAVR
SAVR story reveals how difficult it can be to bring genetically
engineered products to market, how objections with little or no scientific
merit can influence the outcome, and how important public opinion is in
determining commercial success.
Circumstantial evidence available in the 1980s suggested that the tomato
fruit enzyme polygalacturonase (PG), because of its ability to dissolve
cell-wall pectin, was key to fruit softening. Researchers at Calgene, Inc.,
in Davis, proposed to suppress PG accumulation in ripening tomatoes by
introducing a reverse-orientation copy of the gene, an “antisense” copy
designed to prevent or drastically reduce the formation of PG.

Their expectation was that ripe fruit would remain firm longer, perhaps
even allowing it to be transported to market after vine-ripening.
Transporting vine-ripened fruit would avoid the practice of picking
green fruits and artificially ripening them by ethylene treatment, which
gives a ripe tomato color but not the full array of vine-ripened tomato
flavors.
By 1987, Calgene researchers identified and cloned a tomato fruit PG
gene, developed methods for tomato transformation and regeneration,
and produced tomato plants with inserted PG antisense DNA
constructions. Some of the resulting tomato lines generated as little as
1% of the PG found in conventional tomatoes. Based on the results from
eight contained field trials, in October 1992 the U.S. Department of
Agriculture determined that the PG-antisense tomato lines were not a
“plant-pest” risk and no longer required permits for field testing or
transport.
In May 1994, the U.S. Food and Drug Administration, responding to a
Calgene petition, approved the introduction of kanamycin-resistance
gene constructions needed to create the PG-antisense tomato lines.
Kanamycin-resistant organisms in human and animal guts and in soil
were determined to be so common and abundant that they would
overcome any potential influence of the corresponding genes in
engineered crop plants. Allergic reactions to the kanamycin-resistance
protein were also determined to be highly unlikely.
Data submitted by Calgene, including animal feeding studies, showed
the PG-antisense tomato to be indistinguishable in almost every way
from traditional tomatoes. The exceptions were that fruit cell-wall pectin
degraded more slowly, and tomato paste had a higher viscosity.
Paralleling Calgene's efforts to develop the PG-antisense tomato lines,
the company began to gain experience in the conventional fresh-market
tomato business and to meet with community leaders, media
representatives and consumers in Davis and Chicago, the two sites
selected for initial introduction of the FLAVR SAVR tomato. On May
21, 1994, the genetically engineered FLAVR SAVR tomato was
introduced. Demand for this product was high and remained high, but
the product was never profitable because of high production and
distribution costs.
In 1996, Zeneca, under license, introduced in the United Kingdom paste
from PG-antisense tomatoes grown and processed in California, in
collaboration with the grocery chains Sainsbury's and Safeway. More
than 1.8 million cans, clearly labeled as derived from genetically
engineered tomatoes, were sold from 1996 through early 1999. Reduced
processing costs allowed a 20% lower price. The paste from genetically
engineered tomatoes initially out-sold conventional tomato paste at
many locations, but sales of this product declined dramatically in fall
1998. Subsequently, Safeway and Sainsbury's declared that their house
brands would not have genetically engineered ingredients, to satisfy the
stated concerns of some customers rather than for any reason of food
safety.
A report of a select committee of the U.K. House of Commons (1999),
suggests that the decline in sales of the Zeneca tomato paste can be
traced to an August 1998 British broadcast featuring Dr. Arpad Pusztai
and subsequent media attention to the broadcast. He announced his
conclusion that feeding rats genetically modified potatoes resulted in
biological effects that “could” be attributed to the process of genetic
engineering, rather than to the product of the introduced gene (Ewen and
Pusztai 1999). Subsequently, independent analysis of the data,
commissioned by Dr. Pusztai, and his testimony to the select committee
(U.K. House of Commons 1999), both indicate that the conclusions
stated in the broadcast were incorrect. However, the Zeneca product has
not returned to grocery store shelves, with a corresponding loss to
California agriculture.

Marlon project:
Within the EU-funded MARLON project, which ran from 2012-until
2015, we explored the possibility to develop a methodology for post
market monitoring (PMM) of specific potential health impacts of the
consumption of genetically modified (GM) crop-derived feeds by
livestock animals. Under EU legislation, case-specific monitoring may
be required, on a case-by-case basis, by the European authorities as one
of the conditions of marketing approval for such crops.
Whilst such crops have to undergo a rigorous pre-market assessment,
post-market monitoring could serve to verify assumptions or to address
any questions arisen during the previous assessment. Up to now the
requirement for post-market monitoring of GM feed impacts has not
been imposed by the EU authorities for any GM feed yet. With the aim
to assist applicants, risk assessors, decision makers, and veterinary
health professionals with any potential future requirement for the post-
market monitoring of a given GM feed, the MARLON project aimed to
identify the gaps and to develop a generally applicable tool and
methodology as it could not be known on beforehand for which crop
species, which target livestock species and what health effect the
monitoring would be asked for. In the development of a generically
applicable monitoring methodology, two basic questions are asked,
namely: 1) which effects are already known to be associated with the
consumption of GM feeds by livestock; and 2) which indicators of a
potential health impact can be used to monitor for such effects of feeds
in a post-market monitoring program. This review examines four
scenarios of potential health effects and attempts to define health
indicators in the frame of four case studies: allergenicity (CS-1),
horizontal gene transfer (CS-2); mycotoxins (CS-3); and second-
generation nutritionally altered crops (CS-4).
Within the project Marlon, one of the aims was to formulate those health
indicators that can be used to measure possible health impact of GM-
crop-based feed regimes to farm animals; a desk study was performed to
define the potential risk scenarios that could arise for consumption of
GM crop-derived feed ingredients by livestock. The potential risk
scenarios, some of which are already rigorously covered by risk
assessment practices for GM crops are: potential allergenicity, horizontal
gene transfer, nutritionally altered GM crops and the potential positive
health effects deriving from lower mycotoxin levels.
The indicators can be used for the setting up of the epidemiological risk
assessment and dynamic models in order to establish a harmonized
approach towards the post-market monitoring of GMOs used as animal
feed.
 Bibliography
 https://www.sciencedirect.com/science/article/pii/S027869151730
4842
 https://en.wikipedia.org/wiki/Genetically_modified_organism
 https://en.wikipedia.org/wiki/Horizontal_gene_transfer
 https://www.britannica.com/science/genetically-modified-
organism
 http://knowgenetics.org/introduction-to-genetically-modified-
organisms-gmos/
 https://link.springer.com/article/10.1007/s00216-003-1767-7
 https://link.springer.com/article/10.1007/s002170100415
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536854/
 http://calag.ucanr.edu/Archive/?article=ca.v054n04p6