ALTERNATIVES TO ANTIBIOTICS IN SWINE DIETS:

A MOLECULAR APPROACH

Denis O. Krause, James D. House, and C. Martin Nyachoti

Department of Animal Science

University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

Telephone: 204-474-6126
Fax: 204-474-7628
E- mail: denis_krause@umanitoba.ca
SUMMARY

Sub-therapeutic antibiotics are included in animal diets, particularly pig diets, because
they suppress pathogenic bacteria and result in improvements in nutrient absorption by
animals. As antibiotics have become cheaper, the frequency of use in animal feeds has
increased. Unfortunately, the effectiveness of antibiotics to cure infections in humans has
been decreasing. This has raised the concern that using antibiotics in animal feeds has
increased the ability of pathogenic bacteria to develop resistance to antibiotics. For
example, the antibiotic avoparcin, when removed from animal feeds in Sweden, resulted
in a significant reduction in vancomycin resistance in human clinical isolates. More
recently, a 12-month, population-based, case-control study of fluoroquinolone-resistant
Campylobacter infections found that infected individuals were 10-times more likely to
have eaten poultry that had fluoroquinolone in the feed. Although proving a direct link
between antibiotic use in animal feeds and human clinical resistance is difficult, the
epidemiological data are compelling, and irrespective of the scientific evidence,
consumers increasingly demand that meat products be raised antibiotic free, or at least
with the minimum of antibiotics. In Denmark when antibiotics were completely removed
from pig rations, there was an increase in digestive and respiratory tract infections, which
resulted in increased use of therapeutic antibiotics. Ultimately, the total amount of
antibiotics used increased. The challenge is to find alternatives to antibiotics that mimic
their effects.
INTRODUCTION

In animal agriculture, the relationship between host and microbe is becoming

increasingly important given that the routine use of sub-therapeutic (low) levels of
antibiotics in animal feeds is becoming increasingly questionable and has been banned in
other jurisdictions (Yan, 2004). It is also clear that the complete withdrawal of feed
antibiotics have a detrimental effect on production and health, to the extent that more
antibiotics may ultimately be subscribed (Yan, 2004). Alternatives to sub-therapeutic
antibiotics are needed but to make this transition, a more fundamental knowledge about
the role of microorganism in gut function is required (Hooper et a., 2001, 2004)

At present, our understanding of the mechanisms of action of sub-therapeutic antibiotics

can be summarized as follows; (i) inhibition of sub-clinical infections, (ii) reduction of
growth-depressing microbial metabolites, (iii) reduction of microbial use of nutrients, and
(iv) enhanced uptake and use of nutrients through the thinner intestinal wall associated
with antibiotic- fed animals. These share the common postulate that intestinal bacteria,
whether commensal or pathogenic, depress animal growth, either directly or indirectly,
through their metabolic activities (Collier et al., 2003; Deplancke et al., 2002b; Simpson
et al., 2000).

ANTIBIOTICS IN ANIMAL AGRICULTURE

The inclusion of sub-therapeutic (low) levels of antibiotics in animal feed has become a
mainstay of animal agriculture (Gaskins, 2002). Antibiotics were discovered more than
50 years ago. They were initially not include in dietary formulations because of their
cost, which was as high as $200 per kg (Yan et al., 2004). However, by the mid 1960’s
the cost had decreased to a tenth of the original price. Not surprisingly, the level of
inclusion of antibiotics in animals diets has increased in parallel with the decline in cost
from 1 million kg in 1963, to 3 million kg in the 1980’s, to a high of 8 million kg in 2000.
It is therefore not unexpected that there is a growing concern with the ir continued usage
in animal feeds.

The issue is whether sub-therapeutic antibiotic usage in animal feed results in antibiotic
resistance in humans. This question is difficult to address because antibiotics used in
human medicine are generally the same ones that are used in animal feeds (Chee-Sanford
et al., 2001). Given this situation, it is not possible to conclude that there is a direct link
between the two. However, there is a body of related information that would suggest that
the prudent use of sub-therapeutic antibiotics would be advisable.

At the University of Kentucky, a study was performed with swine in which one group of
pigs were not exposed to antibiotics for a period of 28 years (Cromwell, 2002). Over this
period there was a 2.4 % yearly decline in tetracycline resistant fecal coliforms for the
non-antibiotic herd, while those remaining on tetracycline had a virtually negligible
increase in resistance (0.07% increase per year). This study implies that animals given
sub-therapeutic levels of antibiotics in their feed did not show a significant increase in
antibiotic resistance over an extended period of time. Unfortunately, this study, often
used to defend the use of antimicrobials in food animal production, is misleading. In the
Kentucky study only resistance in a minority of cultured bacteria was assessed. The
study did not address the issue of commensal bacteria as a reservoir of antimicrobial
resistance determinants (Chee-Sanford et al., 2001). Additionally, not all antibiotics
show the pattern of resistance found with tetracycline. For example, when avoparcin was
removed from food animals there was reduction in vancomicin resistance in human
clinical isolates (McDermott et al., 2002). While animal usage of antibiotics may not be
the whole story, it certainly is a cog in the machinery of antibiotic resistance.

Most research associated with the transfer of resistance from animals to humans comes
from studies done on Salmonella and Campylobacter infection and “indicator organisms”
such as enterococci and Escherichia coli, which cause disease in humans but not animals.
In the case of resistance acquired in Salmonella and Campylobacter through animals,
there is a lack of data suggesting that this contributes to the burden of human disease
(McDermott et al., 2002). Due to a lack of adequate molecular epidemiological studies,
there is no concrete evidence to prove that resistant enterococci or E.coli from food
animals are even able to colonize the human intestinal tract {McDermott et al., 2002).
There is a lack of clear-cut evidence showing the link between antibiotic resistance and
their use as growth-promoters in animal agriculture. However, having said this it is still
prudent to reduce the use of antibiotics given that a complete picture has yet to emerge as
to the mechanism and evolution of antibiotic resistance.

Consequently, the benefits of using antibiotics far outweigh the known risk of the
development of antibiotic resistance. Antibiotics are effective in improving growth and
efficiency of feed utilization. Data collected from over 1,000 experiments in the United
States from 1950 to 1985 showed an increase in growth rate and feed efficiency in all
phases of growth; with the greatest increase in young pigs. Growth rate increased by
16.4%, 10.6% and 4.2% during the starting phase (7-25 kg ), growing phase (17-49 kg),
and growing- finishing phase (24-89 kg), respectively. Likewise, feed efficiency
increased by 6.9%, 4.5% and 2.2% during the starting phase, growing phase and
growing- finishing phase, respectively (Cromwell, 2002; Yan et al., 2004).

CONSEQUENCES OF ANTIBIOTIC WITHDRAWAL

In order to assess the consequences of antibiotic withdrawal on animal agriculture it is

helpful to assess the effects this has had in European countries which have banned
subtherapeutic use in animal feeds. In 1986, Sweden introduced a general ban on all
subtherapeutic antibiotics in animal feeds. Following this ban there was a two- fold
increase in the incid ence of post-weaning diarrhea (Stein, 2002; Yan, 2004).
Consequently, there was a significant increase in the therapeutic use of antibiotics
because of an increase in disease load in nursery pigs (Stein, 2002; Yan, 2004). In 1988
and 1989, the total use of antibiotics for nursery pigs increased 5-6% compared to 1985
levels (Stein, 2002; Yan, 2004). In 1989, 75% of all nursery pigs were fed diets
containing antibiotics at therapeutic levels. However, the therapeutic use of antibiotics
was partly alleviated by increasing the amount of zinc oxide in nursery diets and by 1995,
only 11% of pigs were fed diets containing antibiotics (Stein, 2002; Yan, 2004).
Withdrawal of subtherapeutic antibiotics from growing- finishing diets had no significant
effect on disease level and the use of antibiotics at therapeutic levels.

By the early 1990’s all countries in the European Union considered banning the use of
avoparcin and by 1999, a ban was introduced for tylosin, zinc bacitracin, spiramycin,
virginiamycin, carbadox, and olaquindox. By 2000, no feed antibiotics were used in
Denmark. Results from antibiotic withdrawal in Denmark were comparable to those in
Sweden showing that omitting growth promoters from the diets of nursery pigs, causes an
increase in sub-clinical and clinical disease, thereby increasing the need for antibiotics at
therapeutic levels (Stein, 2002; Yan, 2004).

In Denmark and Sweden, omitting feed antibiotics from the diet of growing- finishing
pigs decreases performance based on feed efficiency and average daily gain. Therefore,
if the increase in growth rate and feed efficiency for the growing- finishing phase was
4.2% and 2.2%, respectively, with the addition of feed antibiotics in the diet, then these
parameters will decrease by the same amount. However, the impact of removing feed
antibiotics from the diet of nursery pigs is much more severe. There has been an
observed increase in mortality (30%) of nursery pigs which lack feed antibiotics in their
diets. In addition, there has been an increase in total feed consumption from weaning to
25 kg (2-3kg) and a decrease in daily gain (19g/day) (Yan, 2004). Consequently, this has
resulted in a decrease in feed efficiency for this period. Most of the problems associated
with antimicrobial growth promoters withdrawal in nursery pigs can be accredited to
diarrhea caused by E.coli, Clostridium perfringens, and Lawsonia colibacillosis as
previously discussed (McDermott et al., 2002; Yan, 2004).

It has already been established that imprudent use of feed antibiotics is neither desired
nor acceptable in animal agriculture due to the development of antibiotic resistance and
its possible ramifications on human health. However, even in Denmark, where the feed
antibiotics ban is in place, animal strains of non-pathogenic E coli are much less
frequently resistant to antibiotics than strains causing infections in humans;10% in pigs
versus 30-45% in humans (Stein, 2002; Yan, 2004). In addition, animal strains are more
susceptible to antibiotics such as sulphonamides and are never resistant to ciprofloxacin.
These patterns suggest that resistance in E. coli is more likely to be driven by human
antibiotic use, although an animal origin cannot be excluded. Equally as important is the
fact that comple te withdrawal of feed antibiotics has serious consequences on animal
health, growth performance and ultimately, the animal industry. The estimated economic
loss to Danish swine producers resulting from the exclusion of feed antibiotics in the
nursery diet is between 0.5 and 1 USD per pig (Stein, 2002; Yan, 2004).

MICROBIAL ECOLOGY OF THE DIGESTIVE TRACT

Therefore our search for fundamental mechanisms governing homeostasis in the gut
epithelial cells must inevitably include the role of gut microbiota. Microorganisms of the
digestive tract form an important component of the immune defenses against invading
pathogens. From this has flowed the concept of colonization resistance of competitive
exclusion. The best way of explaining this concept is to recount the classical experiments
conducted by Numuri with day-old chicks in the 1970’s (Pessi et al., 1999; Reid and
Friendship, 2002). Young chicks are susceptible to infection by Salmonella, and once
infected at a young age it is economically unfeasible to treat. However, if the ceaca of a
healthy Salmonella free adult chicken, rich in bacteria, is sprayed into the oral cavity of
young chicks, they do not develop Salmonella infections. Thus, the normal microbiota
has competitively excluded or resisted colonization by Salmonella.

The nature of the mechanisms at play is not clear, but the concept of a normal or “good”
microbiota, as opposed to an abnormal or “bad” microbiota has persisted. Some of the
evidence relates to modulation of the balance of microbiota in the gut (Madsen et al.,
2001), and its relationship with immune functions (Shanahan, 2004), and the activation or
even deactivation of Toll- like receptors (Rachmilewitz et al., 2004). It is, however, quite
unquestioned that a healthy balance of microbiota in the digestive tract is essential to
normal healthy function of the host (Hooper et al., 2004; Relman, 2002). To find
alternatives to antibiotics it is necessary to understand what normal healthy microbiota
actually means, and so that we know what members of the microbial community in the
digestive tract have beneficial or adverse effects on the health and nutrition of the pig.
An analysis of production data indicates that antibiotics are most beneficial in nursery pig
diets just after the animals have been weaned (Verstegen and Williams, 2002). This is
understandable given that the animals are undergoing a host of stressors constituting
social, nutritional, and immunological changes.

MICROBIOLOGY OF THE DIGESTIVE TRACT

There is an intimate relationship between the mucosal surfaces of the digestive tract and
microorganisms that populate the intestines (Hooper et al.,2004). This population of
microorganisms can be extensive and as many as 1011 -1012 bacteria are present (Relman,
2002). Estimates are that 90% of our cells are microbial, whereas only 10% of the total
number of host cells is eukaryotic (Hooper et al., 2001). Given this scenario it is
surprising that so little attention has been given to the interactions between host and
microbe

Traditionally, the focus on the monogastric digestive tract has been on the organisms
involved in pathogenesis (Nikkari et al., 2002), and less research has been conducted into
the nutritional consequences of autochthonous microorganisms of the digestive tract. In
contrast, an extensive literature base has been developed surrounding the role of
microorganism in the nutrition of the ruminant animal (Deplancke et al., 2002a). The
problem is further compounded by the fact that almost all the pathogens in the human
digestive tract are found in only six divisions of the bacteriaceae, while there are
currently 30 divisions (Lepp et al., 2004; Relman, 2002).

The recognition that there are as many as 30 divisions in the bacteriaceae has been made
possible by acquisition of ribosomal genes from uncultured bacteria in the gut (Rappe
and Giovannoni, 2003). Extensive ecosystem diversity that remains uncultured has
become a unifying paradigm in microbiology and one of the challenges is to ascribe
function to this vast reservoir of microbiota. It is known that the bacteria adhere tightly
to the mucosal surfaces of the digestive tract, and consequently have a significant
influence on the function of the host (Neish et al., 2000; Strober et al., 2002).

The extent of microbial diversity is really quite extraordinary, and can be appreciated
only within the historical context of molecular analysis of microbial diversity with 16S
rRNA genes. In 1987, when Carl Woese first described microbial diversity within a
ribosomal phylogeny, 11 bacterial phyla were recognized (Rappe and Giovannoni, 2003).
In the ensuing years to 2003 another 14 phyla with cultured representative have been
described. However, there are at least 26 phyla with no cultured representatives. This
only describes the bacteria and a similar situation exists for the fungi, archea, and
protozoa. The situation is even more extraordinary when one realizes that the 16S rRNA
genes are imperfect tools for phylogeny which mask considerable diversity because of
their relative lack of sequence heterogeneity (Nikkari et al., 2002). This is a historical
artifact because the 16S rRNA genes are relatively short, have low sequence variation,
but were originally used because sequencing technology was not what it is today.

CONTRIBUTION OF MICROORGANISMS TO NUTRITION

Microbiota ha ve a significant opportunity to influence the host’s nutritional status

because of the close association between epithelium, mucosal immune system,
microvasculature, and enteric nervous system (Hooper et al., 2001, 2004). In the case of
the ruminant we have a reasonably good understanding of how microbes contribute to the
host nutritional status but this is not the situation with non-ruminants because it is
difficult to assess the effect of individual organisms in a digestive tract where
fermentation is not the prime driver of nutrition homeostasis. One approach is to use
continuous culture systems to investigate the interactions between microorganisms and
nutrients (Rappe and Giovannoni, 2003). An obvious limitation to this is the absence of
various elements such as the mucosa and vasculature of the intestine (Deplancke et al.,
2002a; Ouwehand et al., 2000).

In many situations germ- free animals provide the only mechanism by which the complete
intestinal tissue surface and all its functional components are available for studying the
interactions of gut microbiota (Hooper et al., 2001; Sobko et al., 2004). When
conventionally raised animals are compared to germ free animals there are a number of
anatomical and physiological phenotypes that are collectively referred to as the
microbiota associated characteristics (MACs). Alteration in MACs in conventional and
germ free animals provides a good study in the presence or absence of antibiotics forms a
good case study on the effects of antibiotics on gut function.

A clear difference between germfree and conventional animals is a thinner wall of the
small intestine, with a reduction in connective tissue and lymphoid elements.
Microscopic evaluation of the germfree intestine reveals a more regular and slender villus
structure, with a thinner lamina propria (Gaskins, 2002). Further, the rate of renewal of
epithelial cells is slower in germfree animals, which may have a beneficial effect on basal
energy expenditure and the efficiency of nutrient utilization. These observations are
consistent with the view that in rapidly growing young animals, the GI tract and skeletal
muscle draw from the same limited supply of nutrients and, in effect, compete for
nutrients.

ALTERNATIVES TO ANTIBIOTICS

If the use of antibiotics in animal feed is considered to be unacceptable, then we must

find alternatives which will serve the same purpose of improving animal performance and
maintaining the health status of the animal (Verstegen and Williams, 2002). There are a
number of alternatives which are commonly used in place of antibiotics; these are
probiotics, prebiotcs (non-digestible oligosaccharides), organic acids, enzymes and,
modifiers of microbial activity (herbs) (Pedersen et al., 2004; Verstegen and Williams,
2002). Some of these have been studied in humans, others in animals. Regardless, these
options offer a non-antibiotic approach which may be safer yet still improve animal
performance.

The use probiotics is essentially the addition of a bacterial culture to animal feed (Reid
and Friendship, 2002; Verstegen and Williams, 2002). The mechanism of probiotics
within the digestive tract is prevention of colonization by potentially pathogenic
organisms. However, it has also been discovered that absorption of nutrients through the
gut may also be enhanced through the use of probiotics (Pessi et al., 1999; Reid and
Friendship, 2002). The addition of probiotics derived from fermented dairy products in
human diets has proven effective in reducing signs of lactose intolerance, reducing the
duration of several types of diarrheal diseases, reduction of bacterial enzyme activity and
also may enhance the immune system. The reduction of diarrheal diseases and the
function of the immune system are of particular importance and may be applicable to
swine producers.

Prebiotics or non-digestible oligosaccharides are another effective alternative to the use

of antibiotics (Verstegen and Williams, 2002). The mechanism behind prebiotics is that
they beneficially affect the host by increasing the prevalence or activity of one or a
limited number of commensal bacteria in the intestine, leading to an improvement in host
health (Verstegen and Williams, 2002). Therefore, prebiotics provide a substrate for the
beneficial microbes of the gut. The bacteria present in the intestine of the pig are capable
of fermenting non-digestible carbohydrates (Verstegen and Williams, 2002). Under the
non-optimal conditions imposed on the post-weaned pig, non-digestible oligosaccharides
may have a beneficial effect and be able to control post-weaning diarrhea (Verstegen and
Williams, 2002). In humans, the immunological effects of prebiotics have been studied.
For example, mannose oligosaccharides have been shown to agglutinate pathogens and to
partly function as an antibiotic does (Verstegen and Williams, 2002). In addition, certain
complex carbohydrates provide protection to the host and attach themselves to sites on
the intestinal mucosa thereby preventing adherence of pathogenic microorganisms
(Verstegen and Williams, 2002). In addition, it has been proven that non-digestible
oligosaccharides are able to improve mineral absorption (e.g. calcium and magnesium)
by increasing their solubility in the intestine after fermentation (Verstegen and Williams,
2002).
Organic acids (e.g., citric acid, formic acid, fumaric acid, etc.) have been used in
European countries as an alternative to antibiotics and are considered to be a new
generation of growth promoters. Organic acids not only provide energy but it has been
suggested that they improve protein digestion (Blank et al., 1999; Verstegen and
Williams, 2002). There are 4 general mechanisms behind the growth promoting effects of
organic acids; these are decreased gastric pH, improved pepsin activity, decreased
bacterial growth, and increased nutrient digestibility (Verstegen and Williams, 2002).
Organic acids can influence the microbes of the gut by changing the physical conditions,
making it less optimal for growth of pathogenic species or even intolerable for some
pathogens. For example, a recent study at the University of Manitoba showed that adding
fumaric acid into pig starter diet prevents intestinal colonization with E. coli and
incidences and severity of E. coli associated diarrhea (Owusu-Asiedu et al. 2003).

Exogenous enzymes added to monogastric diets have been reported to have many
beneficial effects, particularly in young pigs. The most important effect is increased
nutrient availability from the diet associated with the use of enzymes in feed. However,
it has been suggested that feed enzymes can exert indirect effects on animal health and
performance by manipulating the growth of both pathogenic and non-pathogenic
gastrointestinal microorganisms (Bedford, 2000). Indeed, recent studies with nursery pigs
(Kim et al., 2003) and broilers (Mathlouthi et al., 2002) suggest that the use of feed
enzymes may positively impact on intestinal health. Also, Inborr and Ogle (1988) in a
study with a limited number of pigs reported that supplementing moistened barley with a
mixture of carbohydrate degrading enzymes was effective in reducing both the incidence
and severity of diarrhea in piglets. The use of feed enzymes as antimicrobial growth
promoters will need to be studied further before this strategy finds widespread use within
the swine industry.

When Europe banned the use of antibiotics in animal feed, it became necessary to find
alternatives in order not to compromise animal health and performance (Verstegen and
Williams, 2002). The alternatives discussed previously are effective in part or entirely in
mimicking the mechanisms of antibiotics. It may be possible to use these alternatives in
combination to achieve the same effect that antibiotics have on pig performance and
health. With ongoing research, it is possible to discover many other potential compounds
which can replace antibiotics in feed. For example, the use of essential oils, conjugated
linoleic acid and herbs and spices are all promising substitutes in the research of
antibiotic alternatives (Verstegen and Williams, 2002).

CONCLUSION

If for no other reason, antibiotic usage in feeds for swine must be curtailed to main access
to markets. In the Western world, the public has an aversion to anything which would
suggest that the safety of the food source has been compromised. One of these is
antibiotics and its presumed link to increasing antibiotic resistance in clinical medicine.
Clearly, from the Scandinavian experience complete removal of sub-therapeutic
antibiotics is not necessarily beneficial. Rather, alternatives must be found that if not
mimic, at least approach the same mechanistic function of antibiotics.
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