WELCOME

PROBIOTICS AND MUCOSAL IMMUNITY

By:Someshwar Zadbuke and Mayank

Tandon

IVRI, Izatnagar, U.P., India and NDRI, Karnal, Haryana, India

mayanktandon1@gmail.com

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

Probiotics Control SE

Initial BW kg

40.0 37.2 1.13

56d BW kg 79.9 73.4 1.85

BW gain kg 40.5 36.2 1.39

Feed conversion

1.59 1.64 .028

Diarrhea, cases

1* 7

Fumiak et al, 1995

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

Performance of WL layers fed 15.3%, 14.3% and 13.8% CP diets containing Lactobacillus CCMS premix

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

(Nahashon et al., 1996a)

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 WL pullets fed corn-soya meal diet with CCMS and Lacto-CCMS premix from 7 to 19 weeks of age (Nahashon et al., 1996b)

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 observed

10 3 6 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 (Sanderson et al. 1993)

Protection- host defense 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 eliminate it”

Innate immunity Adoptive immunity Cells mediating IR:

Lymphocytes Macrophages Epithelial cells Ag presenting cells

(Erikson and Hubbard 2000)

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 Peyer’s patches – covered by M cells

Ag T cells Effector cells

Active immune suppression

Differentiation of IgA secreting B cells

Tolerogenic form

(Sanderson and Walker 1993)

(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”

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

(Strobel and Mowet, 1998)

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

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 activity

0.56 (0.19) 2.25 (0.95)**

*P < 0.05, **P < 0.01

(Strompfova et al. 2005)

Effects of probiotics on nonspecific immunity

Citation Probiotic Species Assessment Effect

Matsuzaki et al. 2000

L. casei Shirota

Mice Innate immune response

Increased NK cell activity from mesenteric node not of PP or spleen

Gill et al. 2000

L. rhamnosus (HN001, DR20), L. acidophilus (HN017), or B. lactis (HN019, DR10)

Mice responses to concanavalin A and lipopolysaccharide

Increased phagocytic activity with elevated γ-INF

Matsuzaki et al. 1995

L. Acidophylus or B. bifidum oral (Live)

Rat Peritoneal or peripheral blood macrophages

Enhanced phagocytosis

Dalloul et al. 2003

Different srains of Lacto-bacillus

Chicken Challenged with Coccidiosis

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 receptorsinteract with epithelial cells – attract other immune cellsunexplored area (Kaila 1992, Isolauri et al. 2001)

Response to Bacteria

BacteriaGram + ve

Peptidoglycan (PG) Muramyl dipeptide (MDP)

Gram - vePeptidoglycan (PG)Lypopolysaccharide (LPS)

Development of immune system by cell stimulation

Receptor dependent process involving cell surface CD 14

Toll receptors associated with pathogens

Transduction pathways

Expression of several immune response genes

Macrophages, Endothelial cells, Smooth muscle cells, Neutrophylls

Mediators – Cytokines

CYTOKINE NETWORK

(Yasui and Ohwaki, 1991, Erikson and Hubbard 2000)

Modualtion of specific immunityCitation Probiotic Animal Assessment Effect

Matsuzaki et al. 1995

L. Casei (Live)

Mice Infection and Ab production in malnourished animals

Increased sIgA and reduced enteric infection

Benyacoub et al. 2005

E. faecium Dog- puppies

Assessment of immune response in post weaning stress

Elevated fecal IgA, circulating IgA, IgG Increased mature B cells No difference in CD4+ and CD8+

Haghighi et al. 2005

L. acidophillus, B. bifidium

Chicken Challenged with SRBC BSA TT

Increased IgMNo responseIncreased IgG, IgA

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

Effect of probiotics on intestinal mucosal immunity and ultrastructure of the cecal tonsils of chicken

(Yurong et al. 2005)

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

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

Benyacoub et al, 2005b

Effect of Bifidobacterium brave, LPS and Con A on the proliferation of PP cells in mice

Mitogen PP cells Unfractionated cells

B cell enriched fraction

T cell enriched 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

(Yasui and Ohwaki, 1991)

Recirculation of MIS (Common mucosal system) ~2% lymphocytes pool recirculating each hr MALT and the systemic lymphoid system Lymphocte cells adhesion molecules - spc for mucosal post

capillary venulePeyer’s patches

Lymphocytes

Regional lymphnode

Mescentric group

Thoracic duct

Blood vascular system

Intestinal lamina propria

Other secreting tissues

(Brandtzaeg et al. 1999)

(Cebra et al. 1991)

Probiotic effects in rodent models of some diseases

Disease model Probiotic Assessment Effect

Insulin dependent diabetes mellitus

L. casei, oral (live)

T-cell markers, splenic cytokines

Decreased CD4+ cells, IFN-γ and IL-2

Imnsulin dependent diabetes mellitus

L. casei, oral (Heat killed)

Splenic T and B cell number and production of cytokines

Decreased incidence of diabetes, increased CD45+ b-cells and IL-2 decreased CD8+ T- cells and IFN- γ

Influenza immunization

B. Bifidus oral

Resp. tract infection and antiinfluenza virus IgG

Protection against lower respiratory tract infections Higher serum IgG levels

(Errikson and Hubbard 2000)

Mean antibody titer against Newcastle disease vaccine on day 42 in chicken

Strain Control OTC L. Casei

HH 2.42b 3.01ab 3.37a

SS 3.19 3.01 3.22

(Zulkifli et al. 2000)

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

Intraluminal Ag

Beneficial/ own

microflora- probiotics

Enhance immune response

Non-beneficial Ag

Adhered to epithelial cells

Entry via enterocyte

Escape tolerance induction of peyer’s patches

promote OT

Balance of metabolic activity and gut microflora

Balance of 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