Probiotics and Prebiotics in Animal Health and Food Safety

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 Caroline Nunes de Almada, Carine Nunes de Almada and
Anderson de Souza Sant’Ana

11 Probiotics and Prebiotics in Animal Health and Food Safety:​

Conclusive Remarks and Future Perspectives
Diana Di Gioia and Bruno Biavati
© Springer International Publishing AG 2018
Diana Di Gioia and Bruno Biavati (eds.), Probiotics and Prebiotics in Animal Health and Food
Safety, https://doi.org/10.1007/978-3-319-71950-4_1

1. Probiotics and Prebiotics: An

Overview on Recent Trends
Georgia Zoumpopoulou1 * , Maria Kazou1 * ,
Voula Alexandraki1 * , Angeliki Angelopoulou1 * ,
Konstantinos Papadimitriou1, Bruno Pot2 and
Effie Tsakalidou1
(1) Laboratory of Dairy Research, Department of Food Science and
Human Nutrition, Agricultural University of Athens, Athens, Greece
(2) Research Group of Industrial Microbiology and Food Biotechnology
(IMDO), Vrije Universiteit Brussel, Brussels, Belgium

Effie Tsakalidou
Email: [email protected]

* Contributed equally

Keywords Probiotics – Prebiotics – Polygastric animals – Monogastric

animals – Aquaculture – Pets – Bees – Applications – Regulation

Georgia Zoumpopoulou, Maria Kazou, Voula Alexandraki, and Angeliki

Angelopoulou contributed equally to this work.

1.1 Introduction
Nowadays, the pro- and prebiotic concept is very well known regarding
human applications related to preserving or restoring health. However,
applications in feed are far less documented. While prebiotics should be
considered a more recent concept, the history of probiotics is long and
interesting. Thousands of years ago, man discovered already the benefits of
“fermented” foods, e.g., in extending shelf life of fresh food products (Ozen
and Dinleyici 2015; Gogineni et al. 2013). This observation was extended
to the fermentation of feed as well. Ancient evidence from Egyptian
drawings and old Carthusian silos shows that more than 1000 years ago,
farmers already knew that silage was an excellent way to preserve summer
crops for their animals during winter times (Mannetje 2010). It took,
however, until the early twentieth century before the Nobel Prize winner
Ellie Metchnikoff for the first time defined and studied the role of the
fermenting bacteria in health (Metchnikoff 1908). While at that time the
“probiotic” concept (live microorganisms that can promote health) was
born, it took until 2001 for the concept to be acceptably defined. This was
achieved by an expert panel composed by the Food and Agriculture
Organization of the United Nations (FAO) and the World Health
Organization (WHO), who defined probiotics as “Live microorganisms that
when administered in adequate amounts confer a health benefit on the host”
(FAO/WHO 2001). Followed by FAO/WHO (2002) and recently revised by
Hill et al. (2014), this definition today is widely accepted by the scientific
community and most governmental institutions.
In the area of animal nutrition, however, in the USA in 1989, also the
term “direct-fed microbials” (DFM) was introduced by the Food and Drug
Administration (FDA). DFM were, similarly as probiotics, defined as “a
source of live (viable), naturally occurring microorganisms.” Clearly, the
health aspect was not maintained in that definition. Consequently, while
manufacturers were required to use this term on their ingredient lists, FDA
did not allow them to make therapeutic claims. FDA together with the
Association of American Feed Control Officials (AAFCO) published a list
of microbial species that could be used in DFM products (AAFCO 1999).
The interest in DFM was already raised in the 1950s when a positive
growth response was noted in animals fed with antibiotics, suggesting that
the suppression of pathogens had a positive impact on the animal health and
improved its growth response. It was not until much later that it was shown
that a healthy intestinal microbiota consists of a certain balance between
potentially beneficial commensal and potentially pathogenic bacteria and
that supplementation with “beneficial” bacteria might evoke a similar
positive growth response, without the need to use antibiotics that left traces
in the meat and caused resistance problems (Landers et al. 2012; Tzivara et
al. 2013). In general, nowadays, two categories of DFM applications can be
distinguished: (1) feed inoculants (e.g., for silage or high-moisture grain),
intended to ferment the feed substrate and modify the digestibility and
safety of the feed component, and (2) beneficial, viable microorganisms
added to feed to improve the health parameters of the consuming animal.
The prebiotic concept is much younger and was first introduced by
Gibson and Roberfroid (1995) as “Non-digestible food ingredients that
beneficially affect the host by selectively stimulating the growth and/or
activity of one or a limited number of bacterial species already resident in
the colon, and thus attempt to improve host health.” As for probiotics, the
original definition has been modified frequently, but so far no consensus
has been reached. Bindels et al. (2015) proposed the definition “A non-
digestible compound that, through its metabolization by microorganisms in
the gut, modulates composition and/or activity of the gut microbiota, thus
conferring a beneficial physiological effect on the host.” In the latter
definition, the “selectivity” is no longer a criterion, and the link with the
metabolic degradation of the compound and the results on the ecology of
the gut microbiota or the observed physiological effects are more deeply
stressed.
Nowadays, in both the pro- and prebiotic definitions, the importance for
health (whether man or animal) occupies a central position. Traditional
applications of probiotics have been mostly performed through fermented
dairy products and were mainly directed to usual digestive processes.
However, more recently other foods and food supplements have gained
much more attention, increasing the worldwide probiotic market, which
exceeded 30 billion dollar in 2015 and expected to account for 52 billion by
2020 (Market Research Report 2016). Moreover, the explosion of the
metagenomics approach to study the microbiota niches in man and animal,
and their exploration in terms of health and disease, has opened a
completely new range of applications, situated in the pharmaceutical field
on top of the nutritional field. The applications of pro- and prebiotics in pet
animals and their exploitation in farm animals, in aquaculture, or even in
plants are equally expanding. The global probiotics in the animal feed
market are expected to attain 4.71 billion dollars by 2021 (Market Report
2016). While the potential of these applications is high at the prophylactic
as well as therapeutic level, there are currently a number of technological,
microbiological, and regulatory bottlenecks, which slow down the
developments in the field and which will be explored further in this chapter.
The technological challenges for probiotics are mainly related to the
requirement that the microorganisms should be viable at the end of the
product’s shelf life. While spray-dried vegetative cells know a faster decline
in numbers over time compared to freeze-dried, the latter is considerably
more expensive, which, for agricultural applications, is a considerable
bottleneck. The use of spore-forming bacteria has, therefore, been proposed
successfully. Spores are better in resisting environmental conditions, such
as heat, moisture, and pH changes. Upon digestion by the animals, they will
germinate into active vegetative cells. Because of their resistance
properties, they are frequently added to pelleted diets, where they survive
the thermal treatments that are often necessary in feed compacting and
pelleting.
The strain selection. Probiotic effects are known to be strain specific
(FAO/WHO 2001); therefore, it is important that strains intended for a
particular application in a particular animal are carefully selected. Selection
criteria will differ depending on the animal species (e.g., farm versus pet
animals) and desired application (e.g., growth promotion versus anti-
infection).
The cost issue. The above research requires a considerable amount of
effort, the cost of which has to be borne by the farmer. As financial margins
in animal husbandry are shrinking, the cost of probiotics does become an
issue. A number of older studies have shown, however, that the
supplementation with a mixture of lactobacilli could improve egg
production and feed efficiency by 3.03 and 7.41%, respectively, in Leghorn
hens, while a large-scale study comprising 101,615 commercial hens
showed an egg production increase from 69.5 to 72.2% with a feed
reduction from 1.75 to 1.69 kg. For further examples in turkey, pigs, and
ruminants, see Ezema (2013). The use of probiotics in pet animals, mainly
for health or animal well-being reasons, however, has a different economic
reasoning and might be more difficult to calculate in this simple way. In
estimating the cost of probiotics in animals, it might also be important to
consider arguments related to the reduction of antibiotic usage. In the
European Union (EU), in 2006, a ban on the use of antibiotics as growth
promoters was introduced (European Parliament and Council 2003). This
ban did reduce antibiotic resistance development but also increased the
general infection rate in husbandry installations (Bywater et al. 2005;
Casewell et al. 2003).
The short life span of, e.g., broiler chicken, reaching slaughter weight
after 5–7 weeks, leaves little time for probiotics to contribute to the
development of a mature immune system, a process, which takes up to
6 weeks. Therefore, rather than being fed with probiotics, hen and broiler
chicks are most often vaccinated against infectious pathogens, such as
Salmonella or a variety of viruses (Breytenbach 1999). Vaccines are mostly
administered via drinking water or spraying. However, given the need for
fast growing (weight gains of over 50 g per day), selected probiotics could
be used for weight gain purpose.
The regulatory aspect for probiotics is complex. In human applications,
different countries allow different levels of health claims. While in the EU
currently no health claims are approved for human applications with
probiotics, except for yogurt in relation to lactose intolerance, the use of
probiotics in animals is well regulated. Manufacturers of probiotics will
need to provide evidence of the identity, safety, and efficacy of the product,
which will be assessed by a committee of experts (European Parliament and
Council 2003). When approved, probiotic products can be labelled and
marketed as “gut flora stabilizers” under the category “zootechnical
additives,” which is one of the five categories of feed additives defined by
this Regulation (EC) No 1831/2003: (1) technological additives, (2) sensory
additives, (3) nutritional additives, (4) zootechnical additives, and (5)
coccidiostats and histomonostats (European Parliament and Council 2003).
In the USA, the Center for Veterinary Medicine (CVM) within the FDA is
responsible for the recognition of safety, effectiveness, labelling, and
distribution of livestock feeds, pet foods, veterinary drugs, and devices. As
mentioned before, the FDA uses the term DFM for probiotics used in
animal feed, “products that are purported to contain live (viable) micro-
organisms (bacteria and/or yeast)” (FDA 2015). For more detailed
information on global regulations, see FAO (2016).
In order to convince the legislator to recognize the benefits of probiotics
at a much broader scale and promote their acceptability by the farmer and
the consumer, the clarification of the mechanisms underlying the beneficial
properties is extremely important. These mechanisms can be more generic
in nature, e.g., the production of organic acids, the reduction of toxic
amines, or more strain specific, e.g., the production of antimicrobials, such
as bacteriocins or hydrogen peroxide (explaining the competitive exclusion
of pathogens, most often observed for probiotics), or the production of
specific enzymes like amylases, lipases, proteases, and glycosidases, which
can assist digestion. Other mechanisms, such as the stimulation of immune
responses are strain specific, may be limited to a specific period during the
development of the animal, and efficacy is mostly depending on the animal
species. Some of these mechanisms are further discussed in this chapter.

1.2 Polygastric Animals

Ruminants, the most widely distributed group of mammals on Earth,
currently add up to about 150 domestic and wild species, while economic
interest lies mainly in the breeding of cattle, sheep, goats, and water
buffaloes. Ruminants are able to assimilate nutrients from low-quality
plant-based feeds, through their digestive tract, which is uniquely designed
and includes, in contrast to other mammals, a four-compartmentalized
stomach consisting of the rumen, the reticulum, the omasum, and the
abomasum. From the physiological point of view, each chamber performs
different processes. The microbial fermentation of the fibers and solid feeds
takes place in the rumen, while the liquids are transferred to the reticulum,
which serves also to the entrapment of large feed particles, regurgitated
subsequently for optimal digestion. In the omasum the liquids are filtered
and various nutrients are being absorbed, and, finally, in the abomasum the
enzymatic digestion of the feed takes place (Hofmann 1989). While the
ruminant gastrointestinal tract (GIT) consists of different niches, the vast
microbial diversity is observed in the rumen, where the microbial
fermentation of the feed is carried out. The rumen microbiome is composed
predominately of bacterial species but also of methanogenic archaea,
flagellated and ciliated protozoa, fungi, and bacteriophages (Chaucheyras-
Durand and Ossa 2014) with populations at a level of 1010 (bacteria), 108
(protozoa), 107 (archaea), and 103 (fungal spores) colony-forming units per
mL of rumen fluid (Deusch et al. 2015).
The optimized ruminal fermentation is essential in supporting health
and productivity in the ruminants, since several physiological parameters of
farm animals are highly correlated with the abundance of various bacterial
members of the rumen microbiome (Jami et al. 2014). Toward this, the
systematic use of antibiotics was gradually adopted as a common practice in
animal husbandry, targeting, inter alia, the beneficial manipulation of
ruminal metabolism. Nevertheless, their rampant use as growth promoters
in animal feed during the last decades gradually raised concerns, not only
for the antibiotic residues in animal products and the emergence of drug-
resistant microorganisms but also for the well-being of the animals
themselves. In recent years, probiotics and DFM are widely used in the
livestock production, especially in the EU, where the use of antibiotics in
this field has been completely prohibited (Landers et al. 2012; Papatsiros et
al. 2013). However, numerous countries, e.g., China, the USA, Australia,
etc., still employ antibiotics in livestock production, and an unprecedented
increase in usage rate during the next decade is foreseen, mainly in
developing countries (Van Boeckel et al. 2015).
The application of probiotics and DFM in ruminant productivity and
health includes treatment of digestive disorders and reduction of gut
pathogens (Wisener et al. 2015), stabilization of the ruminal pH (Chiquette
et al. 2008), enhanced animal performance, increased feed conversion
efficiency and fiber digestibility (Zhang et al. 2015b), improved milk yield
and composition (Ayad et al. 2013; Maragkoudakis et al. 2010), stimulation
of the immune system (Spaniol et al. 2015), treatment of mastitis (Espeche
et al. 2012), and methane mitigation (Alazzeh et al. 2012). The potential of
some probiotics to bind mutagens either present in feeds or formed due to
stress or GIT infections has been recently also reported (Apas et al. 2014).
The vast majority of the applications concern cows and the pre-ruminant
life of calves, whereas the number of respective studies for lambs, sheep,
and goats has increased over the last years. The probiotic preparations are
delivered to ruminants mainly orally, directly, or in the feed. However, the
oral administration may compromise the probiotic efficacy due to the
adverse conditions prevailing in the GIT. For ensuring the stability and
viability of probiotics, the microencapsulation technology has come into
use, providing protection and controlled deliverance of the probiotic
preparation in the GIT (Qi et al. 2011).
An overview of the respective literature reveals the broad applicability
of the well-studied lactic acid bacteria (LAB) as probiotics and DFM in
ruminants. The use of Lactobacillus, Enterococcus, Streptococcus, and
Bifidobacterium species has been reported. Besides LAB, several other
microorganisms have been studied for their probiotic potential in ruminants.
These include lactic acid utilizers, such as Propionibacterium and strains of
Megasphaera elsdenii, as well as other bacteria, such as Escherichia coli,
Bacillus, and fibrolytic Prevotella species (Dhama et al. 2008; Puniya et al.
2015; Rafat and Hussain 2013; Seo et al. 2010). In two recent studies, the
use of cellulolytic Ruminococcus species in buffaloes and reindeers resulted
in the beneficial modulation of their rumen microbiome (Kumar and Sirohi
2013; Praesteng et al. 2013). While most bacterial probiotics are highly
efficacious in pre-ruminant calves, probiotic yeasts and fungi, such as
Saccharomyces cerevisiae and Aspergillus oryzae, respectively, have shown
greater benefits for adult ruminants (Nagaraja 2012). It has been
demonstrated that their use positively influences certain bacterial
populations and the fermentation patterns in the rumen (Pinloche et al.
2013). Furthermore, non-live products from fermentations of probiotic
microorganisms have been efficiently employed in ruminants (Bernard
2015). In a recent study, it has been demonstrated that there was no evident
benefit from the supplementation of live LAB when compared to the
administration of non-live probiotic extracts (Jenkins and Jenkins 2014).
Among the various studies performed, even kefir has been examined as a
probiotic supplement in ruminants, but its administration did not affect
significantly the physiological parameters of the animals (Atasoglu et al.
2010).
The interest for identifying candidate probiotics for ruminants is
gradually focusing on the autochthonous microorganisms from the various
niches of the target animal and toward their ensuing use in the digestive
tract of the animal, which served as the initial isolation source (Fraga et al.
2014; Nader-Macias et al. 2008). For example, comparison of the probiotic
characteristics among isolates from dairy products and animal rumen
revealed that the latter were more tolerant to bile salts and exhibited higher
inhibition against pathogens (Jose et al. 2015). These findings show that the
adaptation of the microorganisms to a specific ecosystem could play a
significant role in the selection of probiotic candidates and that the probiotic
efficacy of selected isolates might depend to some extent on the original
host. Furthermore, the use of rumen inhabitants as probiotics will result in
enhancing the existing beneficial gut microbiota, which seems to be a
milder method of gut microbiome manipulation than introducing
ecosystem-irrelevant microbes (Kumar and Sirohi 2013). Therefore, the
niches of the ruminant GIT constitute a rich and diverse reservoir for
mining potentially novel probiotics (Tellez et al. 2015). The boost in the
development of high-throughput sequencing techniques revealed an
abundance of non-culturable bacteria in the rumen ecosystem in comparison
to data obtained by conventional microbiology (Kim et al. 2011b). The
recent accumulation of metagenomics studies on the rumen microbiome can
provide a vast body of information concerning not only the composition and
the function of the respective microbiota but also its interaction with the
host and its feed (Morgavi et al. 2013).
The concept of using bacteriophages for manipulating certain microbial
populations in ruminants has been also studied (Callaway et al. 2008; Sheng
et al. 2006). Although phages present high host specificity, their efficient
application requires the identification of the bacterial target in the rumen.
To prevent bacterial resistance, the use of phage cocktails is recommended.
In a recent study, a cocktail of designed bacteriophages was successfully
employed in rats as a biocontrol means against the gut pathogen E. coli,
suggesting further testing for possible use in ruminants (Abdulamir et al.
2014). An effective treatment demands the monitoring of the developing
resistance mechanisms, the use of newly isolated phages from the rumen
environment, and even the development of new phages in the laboratories.
Furthermore, the use of isolated lysins instead of whole bacteriophages
could be a promising alternative. However, there are only few data available
about the rumen virome. Recent studies on the rumen bacteriophages and
their interactions with the rumen bacteria constitute an initial attempt to
study the rumen virome in depth, helping to obtain new insights probably
exploitable in the manipulation of the rumen microbiome (Berg Miller et al.
2012; Ross et al. 2013). The detailed characterization of the rumen virome
would be of great significance, since the endemic ruminal phages could
prove to be either a useful tool (Hallewell et al. 2014) or a drawback for the
probiotic interventions in the animals (Kropinski et al. 2012). Additionally,
further research is needed regarding the potential risk associated with the
use of phages in lactating ruminants and the possible contamination of milk
and dairy products. If the adverse effect on dairy manufacturing is
demonstrated, their application could be limited to meat-producing animals.
The application of probiotics and DFM could also play a decisive role
in the mitigation of rumen methanogenesis, since the reduction of the
enteric methane emissions could be attained through the enhancement of
rumen fermentation efficiency and the augmentation of animals’
productivity (Karakurt et al. 2012). The environmental impact of the
ruminant-derived methane is of considerable importance for the
sustainability of livestock, since it is accountable for 25% of the global
methane emissions produced by anthropogenic activities (Buddle et al.
2011). The use of probiotic acetogenic bacteria and yeasts, mainly S.
cerevisiae, for decreasing rumen’s methane emissions has been studied with
promising results (Jeyanathan et al. 2014). Another interesting aspect is the
use of probiotics for controlling specifically the protozoal population in the
rumen, since it has been reported that methanogens found both attached and
inside ciliate protozoal cells are responsible for 9–37% of the enteric
methane production (Jeyanathan et al. 2014; Martin et al. 2010). The
proportional correlation among rumen protozoa and methane emission has
been confirmed using a meta-analysis approach (Guyader et al. 2014).
Recently, the availability of genome projects on rumen methanogens can
provide information about the dominant microorganisms implicated in
methane production, e.g., methanogenic archaea (Leahy et al. 2013),
leading to a more targeted selection of probiotics and DFM.
The use of recombinant microorganisms with probiotic properties in
ruminants has been also documented. The most successful study concerns
the genetically modified bacterium Butyrivibrio fibrisolvens, in which a
dehalogenase for fluoroacetate encoding gene from a Moraxella soil species
was introduced (Gregg et al. 1994). The modified organism was able to
degrade the toxic fluoroacetate present in forage plants. The results were
encouraging since the microorganism survived in the rumen of sheep and
cattle without the loss of the respective gene (Gregg et al. 1998;
Padmanabha et al. 2004). The same species was also used for the creation
of a recombinant xylanolytic strain. A plasmid containing a xylanase gene
from Neocallimastix patriciarum was successfully inserted into Butyrivibrio
fibrisolvens (Xue et al. 1997), and although the modified microorganism
had enhanced capacity for xylan degradation, it failed to persist in the
rumen (Krause et al. 2001). The recent information obtained from various
sequencing projects and databases reveals the abundance of specialized
microorganisms in the rumen. Thus, it would be difficult for genetically
engineered superbugs to fully colonize the ruminal microbial ecosystem and
exert on the host the benefits for which they have been designed (Krause et
al. 2013).
Similar to probiotics, prebiotics, which are nondigestible
oligosaccharides, are also effective in altering the composition and activity
of the microbiome in the GIT, since they constitute suitable substrates for
the enhancement of certain beneficial ruminal microorganisms. However,
the ability of ruminants to catabolize most of the common prebiotic
compounds creates a limitation in the use of prebiotics as growth promoters
in ruminant production. In addition, several nondigestible oligosaccharides
found naturally in plant cell wall are included in feeds normally used in
ruminant rations (Gaggia et al. 2010), making the implementation of
prebiotics in ruminants possibly unnecessary. The administration of
prebiotics seems to be beneficial on very young ruminants, since these
substrates may contribute to the formation of a desirable intestinal
community, which may further improve the performance of older animals
(Uyeno et al. 2015). The advance of rumen-protective technologies
providing shielding from ruminal digestion, such as encapsulation, may
become useful tools for the eventual use of selected prebiotics in ruminant
feed, as it has been reported for probiotics (Mustapha et al. 2013).
Despite the wide applicability of probiotics and to a lesser extent of
prebiotics in ruminant production and the promising results obtained from
various studies, reproducibility issues are raised, since experimental data
acquired are often inconsistent (Uyeno et al. 2015). A wide variety of
factors, such as the growth environment, the animal species and breed, the
age and physiological state of the animal, the diet, the nature of the
probiotic preparation used (e.g., type of microorganism, live culture, or
lyophilized cells), and even its dose, seem to affect the outcomes of
probiotics’ utilization in livestock. Obviously, comprehensive research is
needed for the reliable and viable use of probiotics and prebiotics in
ruminant production.

1.3 Monogastric Animals

Monogastrics are classified as animals having one simple or single-
chambered stomach with the main agricultural species being pigs, poultry,
and horses. The gut microbiota of pigs mainly consists of bacteria, while a
small percentage of archaea mostly Methanomicrobia and Thermococci
have been also identified (Isaacson and Kim 2012; Lamendella et al. 2011).
In the poultry GIT, 13 phyla of bacteria were discovered with Firmicutes,
Bacteroidetes, and Proteobacteria being the more representative ones with
up to 900 and 500 species in chicken and turkey gut, respectively. Of all the
species found, only 117 out of 900 and 69 out of 500 are established genera
of bacteria with the most predominant genera in both chicken and turkey
being Clostridium, Ruminococcus, Lactobacillus, and Bacteroides. Besides
bacteria, the poultry GIT is also inhabited by methanogenic archaea, fungi,
and viruses (Pan and Yu 2014; Yeoman et al. 2012). Furthermore, the horse
GIT is inhabited by bacteria as well, but archaea, fungi, and protozoa are
also present (Daly et al. 2001).
The composition and activity of intestinal microbiota have a crucial
impact on the animal health, growth, and performance as a whole. After the
ban of antibiotics as animal growth promoters in the European Union,
Korea, and Japan, probiotics gained ground as they present a variety of
beneficial effects including, among others, promotion of gut health and
homeostasis (Hou et al. 2015). Costs, however, have been a major
bottleneck for their routine use.
Over the years, probiotics have been used in a number of different ways
in livestock, but in the 1960s, it was demonstrated for the first time that
Lactobacillus strains were able to improve the growth performance of pigs
(Ahasan et al. 2015). The most frequently used probiotics in monogastric
animals are yeasts (Saccharomyces boulardii and S. cerevisiae) and bacteria
(Lactobacillus spp., Enterococcus spp., Pediococcus spp., Bacillus spp.)
targeting the cecum and the colon. The most common benefits of probiotics
in monogastric animals are the increase of body weight, the reduction of the
risk of diarrhea, the improvement of feed efficiency, and diet digestibility
(Ahasan et al. 2015). Furthermore, probiotics have been assigned to play a
significant role in providing supportive care to piglets during their initial
part of life, while probiotics like Enterococcus faecium and Bacillus subtilis
can reduce the concentration of ammonia in the excreta of poultry (Dhama
et al. 2008). There are many microorganisms to be considered as potential
probiotics, but only a limited number of microorganisms seem to satisfy the
necessary criteria.
In order to identify and detect the GIT microbiota from the animal gut
and feces, several techniques have been developed based on biochemical,
microbiological, immunological, and molecular biological features. Among
them, the expansion of high-throughput sequencing techniques exposed the
plethora of non-culturable bacteria enabling the comprehensive
characterization of the intestinal microbiota of poultry and other
monogastric animals (Danzeisen et al. 2011; Kim et al. 2011a). A full
understanding of the intestinal microbiota and the genomic functions of its
members, i.e., microbiome, will lead to the development of targeted
probiotic strains and novel or improved strategies for effective microbiota
modulation (Chambers and Gong 2011; Choi et al. 2015; Pan and Yu 2014;
Umu et al. 2015). Next-generation sequencing studies on broilers and pig’s
gut microbiota shed light on the age-related bacterial diversity revealing the
importance of gut modulation to improve animal health (Kim et al. 2011a;
Mohd Shaufi et al. 2015). Compared to the other monogastric animals,
there is only a limited number of studies characterizing the equine gut
microbiota using culture-independent methods (Daly and Shirazi-Beechey
2003; Hastie et al. 2008; Shepherd et al. 2012; Yamano et al. 2008).
However, as these techniques have been recently developed, the results are
not always reliable (Sachsenröder et al. 2014).
Although the native gut microbiota is commonly used as a pool for
probiotic candidates, the use of genetically modified strains as probiotics in
monogastric animals is of ongoing interest (Sieo et al. 2005). A species
commonly used for genetic engineering in poultry is Lactobacillus reuteri.
A lot of research has been conducted using strains of this species expressing
heterologous genes in a poultry diet with encouraging results on the growth
performance and welfare of animals (Li et al. 2014; Liu et al. 2005, 2007;
Yu et al. 2008). Since genetic engineering approaches have positive results
in poultry, research is currently focusing on genetically modified strains
capable of expressing more than one heterologous genes (Wang et al. 2014).
Apart from poultry, genetically engineered probiotics are also used in pigs
either therapeutically, e.g., in pancreatic insufficiency, or as feed additives
enhancing livestock production (Drouault et al. 2002; Yin et al. 2010).
The idea of using bacteriophages to manage or eliminate zoonotic
bacteria in poultry husbandry has been established as a cost-effective
approach with significant advantages compared to antibiotics. The chicken
gut microbial imbalance frequently caused by broad-spectrum antibiotics is
avoided using host-specific bacteriophages. These bacteriophages are
naturally self-limiting as they replicate only in the target bacteria and only
as long as the bacteria are present (Atterbury et al. 2007). Recently, due to