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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

Performance of calves fed probiotics
Fumiak et al, 1995

The effect of administration of probiotics (Bifidobacrerium pseudolongurn or Lactobacillus acidophilus) on the survival rate of newborn piglets

Probiotics Control SE Initial BW kg 56d BW kg 40.0 79.9 37.2 73.4 36.2 1.64 7 1.13 1.85 1.39 .028

BW gain kg 40.5 Feed conversion Diarrhea, cases 1.59 1*

Performance of WL layers fed 15.3%, 14.3% and 13.8% CP diets containing Lactobacillus CCMS premix
Treatment
CCMS(g/kg) Lacto(Mg/kg) Cp (g/kg)

(Nahashon et al., 1996a)

HEP (%) 88.6 89.5 89.1 88.3 88.1

FC 2.08a 2.10a 2.08a 2.11a 2.17b

DFC 109 112 112 112 114

EM 52.4a 53.1a 53.9b 53.0a 52.5a

0 20 20 20 20

0 0 1100 1100 1100

153 153 153 143 138

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 Control C+CCMS C+CCMS Lacto DFC g 57b 57b 59a BWG g 261b 234c 272a Feed/Gain 6.11b 6.82a 6.07b

Performance of goat kids on supplementation of Probiotics
Parameters A DMI kgw0.75/d (g) CP Dig % CF Dig % Wt gain(g/D) Feed eff. G/Kg Diarrhea cases observed 68.5 76.8 44.1 64.3 7.7 10 Treatment goups B 68.5 75.5 45.7 87.2 5.6 3 C 71.1 80.1 55.2 94.9 5.6 6 D 71.1 82.6 64.3 89.6 5.4 4

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  Protection- host defense
     

(Sanderson et al. 1993)

External secretions Intestinal, genital and bronchial fluids Intestinal flora Epithelial cell membranes Peristalsis, Proteolysis Birth  Adoptive changes  Maturational changes

(Isolauri et al. 2001)

(Perdigon et al. 2001)

Development of immunity
 “Recognition of a foreign material or

pathogen and mounting of a reaction to (Erikson and Hubbard 2000) eliminate it”

 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  Primary Lymphoid Organs
 

(Phillips and Lamm, 1998)

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) 1996)

 Active suppression and Clonal deletion (Toy and Mayer    

Intestinal permeability Soluble Ag’s Particulate Ag Dose dependent

(Isolauri et al. 1993) (Metzler et al. 2005)

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 Leucocyte count (G/l) Heterophils (%) Lymphocytes (%) Monocytes (%) Basophils (%) Eosinophils (%) Phagocytic activity (%) Index of phagocytic activity
*P < 0.05, **P < 0.01

Control group 16.68 (5.43) 16.20 (12.50) 83.80 (12.48) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 8.0 (1.6) 0.56 (0.19)

Experimental group 14.40 (2.54) 26.40 (7.40) 73.40 (7.81) 0.2 (0.4) 0.0 (0.0) 0.0 (0.0) 16.8 (6.3)* 2.25 (0.95)**

Effects of probiotics on nonspecific immunity
Citation Matsuzaki et al. 2000 Gill et al. 2000 Probiotic L. casei Shirota L. rhamnosus
(HN001, DR20),

Species Mice Mice

Assessment Innate immune response responses to concanavalin A and lipopolysaccharide Peritoneal or peripheral blood macrophages Challenged with Coccidiosis

Effect Increased NK cell activity from mesenteric node not of PP or spleen Increased phagocytic activity with elevated γ-INF

L. acidophilus (HN017), or B. lactis (HN019,
DR10)

Matsuzaki L. Acidophylus Rat et al. or B. bifidum 1995 oral (Live) Dalloul et al. 2003 Different srains of Lacto-bacillus Chicken

Enhanced phagocytosis

Elevated CD3, CD4, CD8 and αβTCR Reduced 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 (Kaila 1992, Isolauri et al. 2001) unexplored area

Response to Bacteria
(Yasui and Ohwaki, 1991, Erikson and Hubbard 2000)

Bacteria
Gram + ve
Peptidoglycan (PG) Muramyl dipeptide (MDP)

Gram - ve
Peptidoglycan (PG)

Lypopolysaccharide (LPS)

Development of immune system by cell stimulation Receptor dependent process involving cell surface CD 14
Toll receptors associated with pathogens Macrophages, Endothelial cells, Smooth muscle cells, Neutrophylls

Transduction pathways Expression of several immune response genes

Mediators – Cytokines CYTOKINE NETWORK

Modualtion of specific immunity
Citation Matsuzaki et al. 1995 Benyacoub et al. 2005 Probiotic L. Casei (Live) E. faecium Animal Mice Assessment Infection and Ab production in malnourished animals Assessment of immune response in post weaning stress Challenged with SRBC BSA TT Effect Increased sIgA and reduced enteric infection Elevated fecal IgA, circulating IgA, IgG Increased mature B cells No difference in CD4+ and CD8+ Increased IgM No response Increased IgG, IgA

Dogpuppies

Haghighi et L. Chicken al. 2005 acidophillus, B. bifidium

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
Mitogen PP cells Unfractionated B cell cells enriched fraction

(Yasui and Ohwaki, 1991)

T cell enriched fraction

Control B. Breve LPS Con A

4,529 9,119** 19,588*** 4,815

401 2,376** 9,228*** 83329***

69 815*** 3,643*** 937***

1,299 1,914 2,272* 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

Other secreting tissues

Peyer’s patches

(Brandtzaeg et al. 1999)

Lymphocytes

Intestinal lamina propria
Blood vascular system Thoracic duct

Regional lymphnode
Mescentric group

Probiotic effects in rodent models of some diseases (Errikson and Hubbard 2000)
Disease model Insulin dependent diabetes mellitus Imnsulin dependent diabetes mellitus Influenza immunization Probiotic
L. casei, oral (live)

Assessment
T-cell markers, splenic cytokines Splenic T and B cell number and production of cytokines Resp. tract infection and antiinfluenza virus IgG

Effect
Decreased CD4+ cells, IFN-γ and IL-2

L. casei, oral (Heat killed)

Decreased incidence of diabetes, increased CD45+ b-cells and IL2 decreased CD8+ Tcells and IFN- γ Protection against lower respiratory tract infections Higher serum IgG levels

B. Bifidus oral

Mean antibody titer against Newcastle disease vaccine on day 42 in chicken (Zulkifli et al. 2000)
Strain HH SS Control 2.42b 3.19 OTC 3.01ab 3.01 L. Casei 3.37a 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 microflora- probiotics
Non-beneficial Ag 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

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