WELCOME

PROBIOTICS AND MUCOSAL

IMMUNITY

By:
Someshwar Zadbuke and Mayank
Tandon
IVRI, Izatnagar, U.P., India and
NDRI, Karnal, Haryana, India
Probiotics
 “Living microorganisms, which upon
ingestion in certain numbers, exert
health effects beyond inherent basic
nutrition” (Guarner, 1998)
 First reported by Elie Matchnikoff (1907)
 Term coined in 1965 (Lilly and Stillwell, 1965)
 Lactic acid bacteria (Lactobacilli,
Bifidobacteria and Enterococci)
 Yeasts
 Safe as opposed to antibiotics (Reid 2000)
 Desirable properties
 Survivability in acidic pH of stomach (Dunne et al. 2001)
 Resistance to digestion by bile
 Ability to adhere to intestinal epithelium
 Antimicrobial substances (Kailaspathy et al. 2000)
 Inactivation of procarcinogen (Perdigon et al. 2001)
 Self GRAS (Weese and Anderson, 2002)
Mode of action
 Antimicrobial substance
 Competitive exclusion
 Anticholesterolemic and antilipidemic factors
(Kim and Lee 2005)
 Modulation of the immune system
 Antitumor activity
 Decrease toxic amines and ammonia
 Sparing effect
 Increase GI tone and motility (Dunne et al. 2001)
Effect of probiotics on animal
performance
 Mixed response reported
 Type of strain
 Single or multiple?
 Duration of feeding
 The effect of administration of probiotics
Performance of calves fed (Bifidobacrerium pseudolongurn or
Lactobacillus acidophilus) on the
probiotics
survival rate of newborn piglets
Fumiak et al, 1995

Probiotics Control SE

Initial BW 40.0 37.2 1.13

kg

56d BW kg 79.9 73.4 1.85

BW gain kg 40.5 36.2 1.39

Feed 1.59 1.64 .028

conversion
Diarrhea, 1* 7
cases
 Performance of WL layers fed 15.3%, 14.3% and 13.8% CP diets
containing Lactobacillus CCMS premix
(Nahashon et al., 1996a)
Treatment HEP FC DFC EM
CCMS(g/kg) Lacto(Mg/kg) Cp (g/kg) (%)
0 0 153 88.6 2.08a 109 52.4a
20 0 153 89.5 2.10a 112 53.1a
20 1100 153 89.1 2.08a 112 53.9b
20 1100 143 88.3 2.11a 112 53.0a
20 1100 138 88.1 2.17b 114 52.5a

Performance of WL pullets fed corn-soya meal diet with CCMS and

Lacto-CCMS premix from 7 to 19 weeks of age (Nahashon et al., 1996b)
Diet DFC g BWG g Feed/Gain
Control 57b 261b 6.11b
C+CCMS 57b 234c 6.82a
C+CCMS Lacto 59a 272a 6.07b
 Performance of goat kids on
supplementation of Probiotics
Parameters Treatment goups
A B C D
DMI kgw0.75/d (g) 68.5 68.5 71.1 71.1
CP Dig % 76.8 75.5 80.1 82.6

CF Dig % 44.1 45.7 55.2 64.3

Wt gain(g/D) 64.3 87.2 94.9 89.6
Feed eff. G/Kg 7.7 5.6 5.6 5.4
Diarrhea cases 10 3 6 4
observed

A- Control, B - L. acidophillus, C - S. cerevisiae, D - L. acidophillus + S. cerevisiae

(Mudgal et al. 1995)

The general health score (GHS) of calves per experiment as
affected by treatment with a multispecies probiotic (MSPB, white
bars) or a calf-specific probiotic (CSPB, gray bars) vs. control
treatment (black bars) (Timmerman et al., 2005)
 Introduction to host
defense and immunity
 Gut Mucosal Barrier – Host defense

 Normal digestive functions (Sanderson et al. 1993)

 Protection- host defense
 External secretions
 Intestinal, genital and bronchial fluids (Isolauri et al. 2001)

 Intestinal flora
 Epithelial cell membranes
 Peristalsis, Proteolysis
 Birth
 Adoptive changes

 Maturational changes (Perdigon et al. 2001)

Development of immunity
 “Recognition of a foreign material or
pathogen and mounting of a reaction to
eliminate it” (Erikson and Hubbard 2000)
 Innate immunity
 Adoptive immunity
 Cells mediating IR:
 Lymphocytes
 Macrophages
 Epithelial cells
 Ag presenting cells
Mucosal immune system (MIS)

 GIT, Respiratory tract, Lacrimal, Salivary and Mammary

gland (Phillips and Lamm, 1998)
 Primary Lymphoid Organs
 Thymus
 Bone marrow
 Secondary lymphoid organs
 Spleen
 Lymph nodes (Tizard 1998)
 MALT (Mucosa Associated Lymphoid Tissues)
 IgA
 Immune cells (Cytokines and Chemokines)
(Portal, 2003)
Peyer’s patches
 Dome region
 microfold cells (M cells) (Mc Ghee and Kiyono, 1992)
 Germinal center- B cells change IgM to IgA
 Ag presenting cells (APC) – dendritic cells
and macrophages (Perdigon et al. 2001)
IgA
 S-IgA Dimeric or polymeric bound by join chain “J”
 secretory component (Brandtzaeg, 1995)
 IgA1 – Small Intestine
 IgA2 – Colon
 Part of common MIS (Cebra et al. 1991)
 Level depends on presence of microflora (gram -ve)
 Excretory function (Lamm et al. 1996)
 Synthesis of IgA
 Ag - immune cells - cytokines
 Interaction
 M cells
 Epithelial cell
 Local immune response (Weiner, 1997)
 Ag delivery systems

 Compounds having adjuvant properties

 Incorporation of Ag in particles (Michalek et al. 1994)
Effect of Antigen on MIS
Control of Ag absorption in Gut

 Immune exclusion
 Immune elimination (Sanderson and Walker 1993)

 Peyer’s patches – covered by M cells

Ag T cells Effector cells
Active immune suppression

Differentiation of IgA secreting B cells

Tolerogenic form
(Strober et al. 1998)
Mechanism of IgA in immune exclusion
of Ag (Brandtzaeg P. 2002)
Oral Tolerance
 “Immunogenic hyporesponsiveness to Ag’s
encountered through the enteric route”
(Strobel and Mowet, 1998)
 T and B cell mediated (CD8+ T cells)
 LPS – maturation of T cell precursors (Perdigon et al.
2001)
 Active suppression and Clonal deletion (Toy and Mayer
1996)
 Intestinal permeability (Isolauri et al. 1993)
 Soluble Ag’s
 Particulate Ag (Metzler et al. 2005)
 Dose dependent
Fate of Ag

Ag --- Intestinal wall (Lamina propria and peyer’s patches)

M cells {absorb- apical memb of PP (glucoconjugates) –

coating of Ag (adhesin) and Transport to immune cells}

Lymphoid cells

Immune
response Pathology

(Tizard 1998 and Erikson and Hubbard 2000)

Probiotic modulation of the immune
system
 altering immunogenecity of Ag
 reducing inflammatory mediators
 reversing intestinal permeability and
enhancing degradation of Ag
 diverting gut Ag uptake toward PP
 enhancing mucosal IgA response
 normalizing intestinal flora
Non specific immune response
 Inflammatory response – PMN and
Macrophage
 Ag presenting cells (APC)
 Lymphocytes
Phagocytic activity in blood of Japanese quail after
application of L. fermentum AD1
(Strompfova et al. 2005)

Parameter Control group Experimental group

Leucocyte count (G/l) 16.68 (5.43) 14.40 (2.54)

Heterophils (%) 16.20 (12.50) 26.40 (7.40)

Lymphocytes (%) 83.80 (12.48) 73.40 (7.81)
Monocytes (%) 0.0 (0.0) 0.2 (0.4)
Basophils (%) 0.0 (0.0) 0.0 (0.0)
Eosinophils (%) 0.0 (0.0) 0.0 (0.0)
Phagocytic activity 8.0 (1.6) 16.8 (6.3)*
(%)
Index of phagocytic 0.56 (0.19) 2.25 (0.95)**
activity
*P < 0.05, **P < 0.01
 Effects of probiotics on nonspecific
immunity
Citation Probiotic Species Assessment Effect
Matsuzaki L. casei Mice Innate immune Increased NK cell activity
et al. Shirota response from mesenteric node not
2000 of PP or spleen
Gill et al. L. rhamnosus Mice responses to Increased phagocytic
2000 (HN001, DR20), concanavalin A activity with elevated γ-INF
L. acidophilus and
(HN017), or B. lipopolysaccharide
lactis (HN019,
DR10)
Matsuzaki L. Acidophylus Rat Peritoneal or Enhanced phagocytosis
et al. or B. bifidum peripheral blood
1995 oral (Live) macrophages
Dalloul et Different Chicken Challenged with Elevated CD3, CD4, CD8
al. 2003 srains of Coccidiosis and αβTCR Reduced
Lacto-bacillus oocysts shedding
Control group- more Abs
against recombinant
coccidial Ag in intestinal
secretions
Specific immune response
Ag - M cells

Peyer’s patches
persists in blood

T helper and
inducer phenotype
Max conc

B cell response
IgA and CD8+

Intraepithelial lymphocytes, T cells with γ,δ receptors

interact with epithelial cells – attract other immune cells
unexplored area (Kaila 1992, Isolauri et al. 2001)
 Response to Bacteria
(Yasui and Ohwaki, 1991,
Erikson and Hubbard 2000)
Bacteria
Gram + ve Gram - ve
Peptidoglycan (PG) Peptidoglycan (PG)
Muramyl dipeptide (MDP) Lypopolysaccharide (LPS)

Development of immune system by cell stimulation

Receptor dependent process involving cell surface CD 14
Macrophages,
Toll receptors associated
Endothelial cells,
with pathogens
Smooth muscle cells,
Neutrophylls

Transduction pathways
Mediators – Cytokines
CYTOKINE NETWORK
Expression of several immune
response genes
 Modualtion of specific immunity
Citation Probiotic Animal Assessment Effect
Matsuzaki L. Casei Mice Infection and Ab Increased sIgA and reduced
et al. 1995 (Live) production in enteric infection
malnourished
animals
Benyacoub E. faecium Dog- Assessment of Elevated fecal IgA,
et al. 2005 puppies immune circulating IgA, IgG
response in post Increased mature B cells No
weaning stress difference in CD4+ and CD8+
Haghighi et L. Chicken Challenged with
al. 2005 acidophillus, SRBC Increased IgM
B. bifidium BSA No response
TT Increased IgG, IgA
 Effect of probiotics on intestinal mucosal immunity and
ultrastructure of the cecal tonsils of chicken
(Yurong et al. 2005)

 Probiotics (B. subtilis Bs964, Candida utilis BKM-Y74, L.

acidophilus LH1F) supplemented orally to chicks from
post-hatch to day 3 of age
 Enhanced response to
 Intestinal fluid - IgAat day 7
 PP – IgG, IgM forming cells at day 10 and 7
 Cecal tonsils –
 IgA forming cells at day 7 to 10
 IgG forming cells at day 7
 IgM forming cells at day 4 to 7
 T lymphocytes at day 7
 Increased density of microvilli and length of cecal tonsils
 Decreased efficiency with ageing of the chicken
Total IgA levels in feces of control and probiotic-fed mice on d 0,
7, 14, and 28 of the trial.P < 0.05
Benyacoub et al, 2005a
 Benyacoub et al, 2005b

Specific anti-canine
distemper virus
immunoglobulin IgG
(upper panel) and
IgA (lower panel) in
the plasma collected
at wk 0, 10, 18, 31
and 44 from puppies
fed diets with or
without SF68
 Effect of Bifidobacterium brave, LPS and Con A on the
proliferation of PP cells in mice
(Yasui and Ohwaki, 1991)

Mitogen PP cells Unfractionated B cell T cell

cells enriched enriched
fraction fraction
Control 4,529 401 69 1,299

B. Breve 9,119** 2,376** 815*** 1,914

LPS 19,588*** 9,228*** 3,643*** 2,272*

Con A 4,815 83329*** 937*** 156,423***

*P<0.02, **P<0.01, ***P<0.001

Recirculation of MIS
(Common mucosal system)
 ~2% lymphocytes pool recirculating each hr
 MALT and the systemic lymphoid system (Cebra et al. 1991)
 Lymphocte cells adhesion molecules - spc for mucosal post
capillary venule
(Brandtzaeg et al. 1999)
Peyer’s
Other patches
secreting
Lymphocytes
tissues

Regional
lymphnode
Intestinal
lamina
propria Mescentric
group
Blood
vascular Thoracic
system duct
Probiotic effects in rodent models of
some diseases (Errikson and Hubbard 2000)

Disease model Probiotic Assessment Effect

Insulin L. casei, oral T-cell markers, Decreased CD4+ cells,
dependent (live) splenic IFN-γ and IL-2
diabetes cytokines
mellitus
Imnsulin L. casei, oral Splenic T and B Decreased incidence
dependent (Heat killed) cell number and of diabetes, increased
diabetes production of CD45+ b-cells and IL-
cytokines 2 decreased CD8+ T-
mellitus
cells and IFN- γ
Influenza B. Bifidus Resp. tract Protection against
immunization oral infection and lower respiratory tract
antiinfluenza infections Higher
virus IgG serum IgG levels
Mean antibody titer against Newcastle disease
vaccine on day 42 in chicken (Zulkifli et al. 2000)

Strain Control OTC L. Casei

HH 2.42b 3.01ab 3.37a

SS 3.19 3.01 3.22

Challeges faced by MIS

 Microbial infections
 Route of entry of Ag
 Dietary indiscretions
 Allergies and food intolerances
 Oral antibiotics
 Disruption of lipid and FA metabolism
 Ageing
 Inflammation – level of IgA
Balance of gut microflora
Intraluminal Ag
Beneficial/ own Non-beneficial Ag
microflora- probiotics
Adhered to epithelial
cells
Enhance immune
response
Entry via enterocyte

promote OT
Escape tolerance
induction of peyer’s
patches
Balance of metabolic
activity and gut
microflora
Choosing probiotics
 Type of immune cells stimulated (Inflammatory or
specific immune response)
 Most active strain
 Dose required for maximum effect?
 When to be administered?
 Is it safe to use probiotics in immunosuppressed
host?
 Storage quality
Future prospects
 Species identification and their use needed to
be quantified
 Identification of direct cause and effect
needed
 Reduced inflammation either at local or
systemic level?
 Can they be used as adjuvants for oral
immunisation?
 Modulation of MALT or a systemic immune
response?
Conclusions
 No clear cut evidence observed on the effect
of probiotics on production aspect of animals
 Competitive exclusion is beneficial in early
phase of life to prevent diarrheal occurrences
 Probiotics modulate immunity of host through
enhanced mucosal immunity (non specific as
well as specific)
 Can be used as prophylactic measures to
enhance health status of the animals