University of duhok

College of veterinary medicine

Probiotics
And their application in Veterinary
Medicine

By
Jivan.Q.Ahmad

2009-2010
Content
1- What are probiotics………………………………..………………..3
2- History of probiotics………………………….…………………….3
3- Mechanisms of action…………………………..…………...………4
3.1.1. Modification of intestinal micro biota……………….......4
3.1.2. Enhancement of barrier function……………....………….5
3.1.3 Immunomodulation………………………………………...6
3.1.4 Competitive exclusion……………………………..………7
3.2. Action of yeast as probiotics ……………...………………. 9
4- Probiotic-criteria……………………………….…………………..9
5- Microorganisms used as probiotics………….……………….…..10
6- Application in animal health………………………………...……10
7- Application in animal production………………………………...13
 Probiotics

The term probiotic is a Greek word meaning “for life” in contrast to antibiotics
which are against life. Probiotics can be defined as “living microorganisms
(bacteria or yeasts) that, when administered orally in adequate numbers provide
a health benefit to the host (FAO/WHO 2001).

History of Probiotics
The original observation of the positive role played by some selected bacteria
is attributed to Elie Metchnikoff 1907 , the Russian born Nobel Prize
winner working at the Pasteur Institute at the beginning of the last center ,who
suggested that" The dependence of the intestinal microbes on the food makes
it possible to adopt measures to modify the flora in our bodies and to replace
the harmful microbes by useful microbes".

At this time Henry Tissier, a French paediatrician, observed that children with
diarrhea had in their stools a low number of bacteria characterized by a peculiar,
Y shaped morphology. These “bifid” bacteria were, on the contrary, abundant in
healthy children. He suggested that these bacteria could be administered to
patients with diarrhea to help restore a healthy gut flora. In 1965, the term
„probiotics‟ was first used by Lilly and Stillwell in a different context to
represent „substances secreted by one organism which stimulate the growth of
another‟

Parker in 1974 suggested an interaction between microorganisms with the host:

“Organisms and substances with beneficial effects for animals by influencing
the intestinal microflora.”
Fuller in 1989 defined it as “A live microbial feed supplement which
beneficially affects the host animal by improving its intestinal microbial
balance.”
Havenaar and Huis Int Veld in 1992 said probiotics are “ A mono- or
mixed culture of live microorganisms which , applied to animal or man , affect
beneficially the host by improving the properties of the indigenous microflora.”
ILSI (International Life Sciences Institute) EuropeWorking Group (1998) :
“A viable microbial food supplement which beneficially influences the health of
the host.”
Diplock et al. in 1999 puts it as “Probiotic food is functional if they have been
satisfactorily demonstrated to beneficially affect one or more target functions in
the body beyond adequate nutritional effects, in a way that is relevant to either an
improved state of health and well-being and/or reduction in the risk of diseases.”
Naidu et al. in 1999 said “A microbial dietary adjuvant that beneficially affects
the host physiology by modulating mucosal and systemic immunity, as well as
improving nutritional and microbial balance in the intestinal tract.”
Tannock in 2000 observed that long-term consumption of probiotics was not
associated with any drastic change in the intestinal microbiota composition, and
thus proposed an alternative definition: “Microbial cells which transit the GI tract
and which, in doing so, benefit the health of consumer.”
Schrezenmeir and de Vrese in 2001 defined probiotics as “ A preparation of
a product containing viable, defined microorganisms in sufficient numbers, which
alter the microflora ( by implantation or colonization ) in a compartment of the
host and by that exert beneficial health effects in this host.”
FAO / WHO ( Food and Agriculture Organization and World Health
Organization) (2001) and Reidet al.(2003) concentrated exclusive Lyon its
health purpose:“Live Microorganisms which when administered in adequate
amounts confer a health benefiton the host.”

Mechanism of action
I. bacteria

 Antimicrobial Activity
 Enhancement of Barrier Function
 Immunomodulation
 Competitive exclusion

Antimicrobial activity

Probiotic bacteria can antagonize pathogenic bacteria by reducing luminal

pH, inhibiting bacterial adherence and translocation, or producing
antibacterial substances and defensins. One of the mechanisms by which
the gut flora resists colonization by pathogenic bacteria is by the produc-
tion of a physiologically restrictive environment, with respect to pH,
redox potential , and hydrogen sulfide production . Probiotic bacteria
decrease the luminal pH, the probiotic B. breve produced a high concen-
tration of acetic acid, consequently lowering the luminal pH. Production
of antimicrobial compounds, termed bacteriocins , by probiotic bacteria is
also likely to contribute to their beneficial activity. Several bacteriocins
produced by different species from the genus Lactobacillus have been
described. The inhibitory activity of these bacteriocins varies ; some
inhibit other lactobacilli or taxonomically related Gram-positive
bacteria, and some are active against a much wider range of Gram-
positive and Gram-negative bacteria as well as yeasts and molds. For
example, the probiotic L.salivarius subsp. salivarius UCC118 produces
a peptide that inhibits a broad range of pathogens such as Bacillus ,
Staphylococcus , Enterococcus , Listeria, and Salmonella species.
Lacticin 3147, a broad-spectrum bacteriocin produced by Lactococcus
lactis subsp., inhibits a range of genetically distinct C. difficile isolates
from healthy subjects and patients with IBD. A further example is the
antimicrobial effect of Lactobacillus species on Helicobacter pylori
infection of gastric mucosa , achieved by the release of bacteriocins and
The ability to decrease adherence of this pathogen to epithelial cells.
Probiotics can reduce the epithelial injury that follows exposure to E. coli
O157:H7 and E. coli O127:H6. The pretreatment of intestinal cells with
lactic acid-producing bacteria reduced the ability of pathogenic E. coli to
inject virulence factors into the cells or to breach the intracellular tight
junctions . Adhesion and invasion of an intestinal epithelial cell line by
adherent invasive E. coli isolated from patients with CD was substantially
diminished by co- or preincubation with the probiotic strain E. coli Nissle
1917. These findings demonstrate that probiotics prevent epithelial injury
induced by attaching-effacing bacteria. Defensins are antimicrobial
peptides involved in innate defense mechanisms . The probiotic E. coli
Nissle strain induced expression of human beta-defensin (hBD-2) in
intestinal epithelial cells; this type of effect may contribute to an
improved mucosal barrier and provide a means of limiting access of
enteric pathogens.

Enhancement of barrier function

In addition to the inhibition of growth of “conventional” organisms or

potential pathogens , probiotics can influence mucosal cell – cell
interactions and cellular “ stability ” by the enhancement of intestinal
barrier function through modulation of cytoskeletal and tight junctional
protein phosphorylation . Intestinal barrier function is maintained by
several interrelated systems including mucus secretion,chloride and water
secretion, and binding together of epithelial cells at their apical junctions
by tight junction proteins. Disruption of epithelial barrier function is seen
in several conditions including IBD , both active and inactive , in the
healthy relatives of patients with IBD, enteric infections, coeliac disease,
And some autoimmune diseases such as Type 1 diabetes. Enhancement
of mucosal barrier function may be an important mechanism by which
probiotic bacteria benefit the host in such diseases . The mechanisms by
which probiotics bacteria enhance gut mucosal barrier function are
unclear , but may relate to alterations in mucus or chloride secretion or

changes in tight junction protein expression by epithelial cells . Some

probiotic bacteria modify MUC gene expression and mucus secretion. For
example , L. plantarum 299 v increased MUC2 and MUC3 mRNA
expression when incubated with the epithelial cell line HT-29.57 VSL#3
and E. coli Nissle strain increased MUC2 , MUC3 , and MUC5AC gene
and protein expression . Some probiotic bacteria limit chloride and water
secretion.
For example, S. thermophilus and L. acidophilus reversed the increase
in enteroinvasive E. coli-induced chloride secretion by an epithelial cell
line. Tight junction proteins are dynamic structures subject to structural
changes that dictate their functional status . In epithelial cells the tight
junction protein zonula occludens-1 (ZO-1) redistributes when exposed
to pathogenic bacteria such as S. Dublin.
VSL#3 , which consists of 4 strains of Lactobacillus (L. acidophilus,
L.casei, L. plantarum, L. delbrueckii) , 3 strains of Bifidobacterium (B.
infantis, B. longum, B. breve ) , and 1 strain of Streptococcus salivarius
subsp.

Immunomodulation

The intestinal tract is a very important part of the immune system. Here is
the first line defense against environmental attacks from pathogenic
bacteria, viruses, and other foreign substances. Total mucosal surface area
of the intestinal tract is around 3,000 ft2, or about the size of a tennis
court. This is a very large surface interface with the external environment.

One of most important effect of probiotic bacteria on epithelial cells

is the ability of commensal organisms to act through pattern recognition
molecules or Toll - like receptors (TLR) , such as TLR-2 and TLR-4,
possibly on epithelial cells . Such interactions induce the production of
protective cytokines that enhance epithelial cell regeneration and inhibit
epithelial cell apoptosis.

The signaling pathways that allow epithelial cells to distinguish

commensal or probiotic organisms from pathogenic organisms appear to
be different . Pathogenic bacteria induce proinflammatory responses in
intestinal epithelial cells by activating the transcription factor NF-_B. In
contrast, nonpathogenic species can attenuate proinflammatory responses
By blocking the degradation of the counter-regulatory factor I_B. This
method of blocking proinflammatory responses is shown by nonpath-
ogenic Salmonella pullorum , which attenuates IL-8 secretion elicited by
pathogenic S.typhimurium . Also there is modulation of humoral immu-
nity by realeasing different antibodies from B cells , for example
L.rhamnosus GG administered to children with acute gastroenteritis
enhanced a nonspecific humoral immune response , reflected by an-
increase in IgG , IgA, and IgM secretion from circulating lymphocytes.
The effects of probiotic bacteria on B lymphocytes and antibody produc-
tion have also been evaluated in vaccination Trials . The immunogenicity
of rotavirus vaccination was enhanced in children who received L. casei
GG compared with those who received placebo.Similarly , increased
Salmonella – specific IgA levels were found in subjects who received a
Combination of L. rhamnosus GG and Salmonella vaccination. L.casei
can enhance NK cell activity ,and Phagocytosis was enhanced by using
L. acidophilus , B. bifidum , or L. johnsonii.

Competitive exclusion

This natural antipathogen activity has been described as „bacterial ant-

agonism ‟ , „bacterial interference‟ , or „CE‟. CE technology involves the
addition of a nonpathogenic bacterial culture of a single strain or multiple
strains, to the intestinal tract of food animals in order to reduce coloniza-
tion or decrease populations of pathogenic bacteria in the gastrointestinal
tract. Typically, CE cultures are isolated from the animal species that will
be treated in order to take advantage of a synergistic interaction between
the host animal and its native microbial ecosystem that developed during
millions of years of co-evolution.
The introduction of a stable, mixed microbial consortium to the naı¨ve gut
of a neonate can aid in the early establishment of a normal microbial
population that can competitively prevent the establishment of a
pathogenic bacteria population . This is especially critical in the poultry
industry as chicks can be quickly colonized by pathogens after hatching.
However , when CE is used in older animals , it must often compete
against an established pathogen population that must be displaced.
Therefore, the mixture of bacteria chosen for use as a CE culture must be
specific for the animal, production stage, and scenario in which it will be
utilized.
CE has been suggested to have several modes of action that eliminate
pathogenic bacteria, including:
 direct and indirect competition for nutrients.
 competition forphysical attachment sites.
 production of antimicrobial compounds (including VFAs).
 enhancement of host immune system activity.
 a synergistic interaction of two or more of the above activities.

If bacteria (including pathogens) cannot grow at least as fast as the

passage rate of their environment , then the constant flow of digesta will
„ wash out ‟ the pathogen . After a CE culture (or the natural flora) is
established within the gut , bacteria bind to the surface of the intestinal
epithelium .
preventing opportunistic pathogens from attaching and thus obtaining
a colonization foothold.VFAs produced by normal microbial fermentation
in the gut are toxic to some pathogenic bacteria, and may reduce the com-
petitive fitness of these bacteria in the gut environment.
II. yeast action as probiotics
Aqueous extracts prepared from Saccharomyces cerevisiae stimulated the
growth of certain rumen micro-organisms .Yeast has been shown to
provide vitamins (especially thiamin) to support the growth of rumen
fungi. High dicarboxylic acids, particularly malic acid, content of the
yeast has also been shown to be the possible cause of in vitro, but it does
not appear to cause the most important effects of yeast in vivo. Removal
of oxygen, which would inhibit the growth of the strictly anaerobic
bacteria of the rumen, was also suggested. Rumen contents are essentially
anaerobic, but low concentration of dissolved O2 can be detected during
the daily feeding cycle. O2 enters the rumen while the animal is eating,
both with the feed and the saliva. The increase in redox potential
observed after the meal in sheep, is mainly due to the supply of oxygen in
the rumen during feed intake, mastication and water intake. The ability of
different strains of Saccharomyces cerevisiae to stimulate the viable
count of bacteria in the rumen appears to berelated to their ability to
remove oxygen from rumen fluid, since respiration-deficient mutants of
Saccharomyces cerevisiae failed to stimulate bacterial numbers .

In monogastric animals involved in stimulation of brush border

disaccharidases, anti-adhesive effect against pathogens, stimulation of
non specific immunity, toxin action inhibition, and antagonistic effect
against pathogenic micro-organisms. Stimulation of brush border
disacharridases have shown that oral ingestion of Saccharomyces
cerevisiae by human volunteers and weaned rats resulted in marked
increases in the specific and total activities of brush border membrane
disacharridases including sucrase, lactase and maltase. This property
could be interesting since some diarrhoeas are associated with a decrease
of the intestinal
disacharidase activities. concluded that increased disaccharidase activities
could be mediated by endoluminal release of polyamines (spermine and
spermidine) produced by live yeast.

Probiotics – criteria
A summary of conventional criteria that can be used for the selection of
microbial strains to be used as probiotics includes the following
properties:

• Biosafety: the strains of microorganisms should be Generally

Recognized As Safe (GRAS microorganisms), for example, Lactobacillus
species or some Bifidobacterium and Streptococcus (Enterococcus)
species.
• The choice of the origin of the strain: probiotics should preferentially
originate from the target animal microflora. This choice is determined by
the specific purpose of the application of the probiotics (e.g. location
specificity or requirement for colonization). The strains should be
properly isolated and identified before use.
• Resistance to in vivo/vitro conditions: after administration
of the probiotic, the microorganisms should not be killed by the defense
mechanisms of the host and they should be resistant to the specific
conditions occurring in the body. They should be resistant to the pH, bile
and pancreatic juice conditions.
• Adherence and colonization of intestinal epithelium/tissue:
factors that affect colonization should be considered. These should
include the resistance of bacteria themselves, the effect of gastrointestinal
environment (ingredient, pH, bile, salt, etc.) on colonization, the existing
microbes that exert interacting factors (probiotics- host-microflora
interactions), etc.
• Antimicrobial activity/antagonisms to pathogens:
Lactic acid bacteria, which are frequently used as probiotics, have a
number of antagonistic properties which operate by decreasing pH by the
production of lactic acid, consumption of available nutrients, decreasing
the redox potential, production of hydrogen peroxide under aerobic
conditions, production of specific inhibitory components, such as
bacteriocines, etc., and which would help to protect against pathogenic
organisms. This is important for the probiotics to be effective.
• Stimulation of immune response
• Viability/survival and resistance during processing
(e.g. heat tolerance or storage).

Microorganisms used as probiotics

 A. bacteria

The crucial event in the development of the probiotic approach to Anima

health was the finding that the newly hatched chicken coul be protected
against salmonella colonization of the gut by dosing it with a suspension
gut content prepared from healthy adult chicken Microorganism used in
probiotic include those derived from the Lactobacillus, Streptococcus,
Enterococcus , Bacillus ,Clostridium, Bifidobacterium species and E.coli
Nissle 1917.
in fish Micrococcus luteus and Pseudomonas species are most commonly
used.

B. yeast

Yeasts and fungal probiotic such as(Saccharomyces cerevisiae) and

amaferm (Aspergillus oryzae) in adult ruminants.

the combination of probiotic strains could increase the beneficial health

effects as compared to individual strains because of their synergistic
adhesion effects.

Application in animal health

1. Probiotic activity is largely inhibitory since the probiotic bacteria
can reduce the level of E.coli O157 carriage and fecal sheding in
cattle and calves.
2. the growth of Enterohemorrhagic Eschericia coli EHEC O157:H7
and production of shige-liketoxins is inhibited by co-incubation
with Clostridia butyricum .
3. treat ruminal acidosis by yeast. probably due to lowering of redox
potential in the rumen, increase in cellulolytic bacteria , and
general improvement in ruminal digestion .

4. probiotics reduce gastric inflammation and bacterial colonization in

Helicobacter pylori - infected animals.And induce inflammatory
response in feedlot steers fed high-grain diets.
 5. Probiotics(lactobacillus spp) can alter balance of gastrointestinal
microflora in healthy cat and are shown to be effective in
preventing antibiotic associate diarrhea.
6. In horses, after antibiotic treatment, stress conditions etc. Safe for
weaned foals and pre-weaned foal's for maintaining proper
digestive balance.
7. Probiotics were widely reported to reduce the incidence of
antibiotic-associated diarrhea the probiotics were used singly (L.
rhamnosus GG, Saccharomyces boulardii) or in combination (L.
acidophilu sþbifidobacteria or Streptococcus thermophilus).

8. Lowering cholesterol levels, presumably by breaking down bile in

the gut, thus inhibiting its reabsorption (which enters the blood as
cholesterol).

In fish

The use of probiotics can improve the nutrition level of aquacultural

animals and improve immunity of cultured animals to pathogenic
microorganisms. In addition, the use of antibiotics can be reduced and
frequent outbreaks of diseases can be prevented. M luteus and
Psuedomonas sp. as growth promoters and antibacterials for Nile tilapia
fry (O. niloticus) and their effect on some physiological parameters.
Streptococcus faecium improved the growth and feed efficiency of Israeli
carp . A several probiotic species were used including Lactobacillus sp.
And could reduce fish mortality challenged with aviriolent strain of
Aeromonas salmonicidae .

Uses in poultry

The bacterial flora of chicken is almost constant throughout the length of

gut and it‟s mainly composed of E.coli, Clostridium spp, Lactobacillus
spp and several other anaerobes. Salmonella spp infection is serious
disease of chickens and also potential human risk. Probiotics have been
shown to be involved in protection against a variety of pathogens in
chicken including Escherichia coli ,Salmonella, campylobacter,
clostridium and Eimeria. The pediococcus acidilactici based probiotic
effectively enhance resistance of birds and partially protect against
negative growth effects associated with coccidiosis. As growth
promoters, probiotic supplementation of chicken diet has been variable
but s,ome papersreport statistical significant effect on growth and egg
production,nitrogen retention and lower cholesterol with Lactobacillus
spp. Broiler also showed an increase in weight gain and better feed
conversion when they were fed with Lactobacillus acidophilus in
drinking water Also reduce leg weakness.

Applications in animal production

I. Improved growth rate

In animal nutrition microorganism used as probiotics was linked

with proven efficiency on gut microflora ,administration of
probiotics strains separately and in combination was significantly
improve feed intake,Feed conversion rate, daily weight gain and
totalybody weight in chicken,pig,sheep,goat,cattle, and equine..
Probiotics has positiveeffect on varios digestive processes ,specialy
cellulolysis and synthesis of microbial proteins. Probiotic are
stabilizerof ruminal ph and lactate, increase absorption of some
nutrients.

II. Milk production

Probiotics have beneficial effect on milk yields, fat and protein

contents. Such in dairy cow when aspergillus oryzae and
Sacchromyces serevisiae have increased milk production, milk
solids-not fat(NSF) increase milk protein percentage,this is due to
the no of cellulolytic bacteria , fiber degration and changes in
volatile fat acid(VFA) in the rumen.

III. Meat production

Probiotics effect on animals by increasing their productivity and

quality of production, also increase carcass output and water
holding capacity and decrease cooking loss and meat hardness.
 Several p strains have been utilized for fermented sausage such as
lactic acid-producing mainly belong to genera
lactobacillus,pediococcus and streptococcus this for production
of consistent and controlled acidification that inhibit growth of
undesirable microorganisms.Therefore in meat industry lactic acid
bacteria are widly used as starter culture for suppression the growth
of S.aureus in the manufacturing of fermented meat products.

IV. Egg production

Probiotics increase egg production and their quality and decrease

egg contamination, also increase egg shell weight, shell thickness
and serum calcium.Also significantly reduce plasma cholesterol
level and triglyceride.
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