swine plasma immunoglobulins for treatment of … · swine plasma immunoglobulins for treatment of...

Swine plasma immunoglobulins for treatment of post-weaning diarrhoea

by Chris Juul Hedegaard, PostdocTechnical University of Denmark

National Veterinary Institute Division of Vaccinology and Immunology

Peter M. H. HeegaardInnate immunology Group

18 August 2016DTU Vet, Technical University of Denmark

Contents

•Postweaning diarrhoea and antimicrobials

•Passive immunisation with porcine plasma purified immunoglobulins (as an sustainable alternative to antibiotics and dietary zinc)

•In vitro and in vivo studies

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Postweaning Diarrhoea

3

From J.M. Fairbrother (2011): E. coli associated Post-weaning diarrhoea. Etiology, clinical signs and risk factors.

• Common condition in weaner piglets (~3 days after weaning)

• In EU: up to 23% of piglets are estimated to be affected

• Usually caused by intestinal colonisation of F4+/F18+ Enterotoxigenic E. coli (ETEC)

• Results in decreased average daily weight gain

• Mortality up to 25% if untreated

Common treatment/prevention: orally administered antibiotics and dietary zinc-oxide containing feed

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Diarrhoea

4

Antibiotics

8

Antibiotics

9

Antibiotics

10


Threat of microbial antibiotic resistance

14

USA (CDC 2013):

Europe (ECDC/EMEA 2009):

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Sales of veterinary antimicrobial agents in 26 EU/EEA countries

15

5th ESVAC report 2013

PCU = animalpopulation correction unit

11/01/201711 January, 2017From: WHO 2012


Antimicrobial consumption in Denmark

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18 August 2016DTU Vet, Technical University of Denmark 11/01/201717

30% of total Danish antibiotics goes to PWD

treatment

Antimicrobial consumption in Denmark

11 January, 2017


Dietary zinc oxide has a negative effect on the environment

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18 August 2016DTU Vet, Technical University of Denmark 11/01/201719

Alternatives to antibiotics are (also) needed in swine production

11 January, 2017

18 August 2016DTU Vet, Technical University of Denmark20

• Leads to future protection (memory)

Active immunisation (Vaccination)

1. As weaner piglets have immature immune system

2. Because of too short time to develop an immune response/protection after weaning

3. Due to interfering (lactogenic) maternal antibodies

4. As live oral vaccine can not be combined with antibiotics

5. If multifactorial

But may not be applicable for preventing enteric diseases:

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• Gives immediate protection/alleviation

• Sow lactogenic immunity surrogateBut no long-term protection (no memory)

Passive immunisation

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Invasion

Immune-responses

Antibodies

Protection

Infectious agents

Acquired immunity

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Blood is slaughterhouse waste X

Slaughter

Inexpensively, untapped and renewable resources

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24

Purification

25

PurificationWash step

Elution

26

Light chain

Heavy chain

Immunoglobulin

Purified Pig IgG = ppIgG

Natural antibodies against Postweaning dirrhoea

Hedegaard et al (2016) PLoS One


ppIgG for prevention and treatment of PWD

Weaner piglet

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Reactivity against bacteriaIndirect (whole-cell) ELISA

0.11100

1

2

3

Y. ruckeri

no IgG

S. DiarizonaeE. coli O138E. coli F4+E. coli F18+

Swine IgG (mg/ml)

Net

OD

450-

650

TMB

Bacteria Bacteria Bacteria Bacteria

= ppIgG

= HRP:anti-pig

IgG antibody

Competitive ELISA

E. coli

0.1 1 100

20

40

60

80

100

***

***

***

*********

***

*** ******

**

mg/ml

% In

hibi

tion

S. Diarizonae

0.1 1 100

20

40

60

80

100Swine IgG60°C70°CPBS

***

***

*** ***

******

mg/ml

% In

hibi

tion

TMB

= ppIgG

= HRP:anti-E. coli/Salmonella antibody

= Bacteria lysate

Hedegaard et al (2016) PLoS One 11/01/2017 31

Reactivity against bacteria

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Indirect ELISA

= Bacteria lysate

TMB

= ppIgG

= HRP:anti-pig

IgG antibody


Litter mix Weaning

0Day 12-28In

ocu

lati

on

1 2In

ocu

lati

on

Ino

cula

tio

n

Ino

cula

tio

n

ppIgG

(4 gr/day)

Autopsy

Faecal samplingNoninfected

Infected

Infected+ppIgG

PWD: experimental model

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34

Shedding of ETEC (faeces)

Enterobacteriaceae in ileum

(p=0.0017)

(p=0.001)


PWD: experimental model

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18 August 2016DTU Vet, Technical University of Denmark 11 January, 201735

Pre-clinical trial (Feacal score)

Litter mix Weaning

Day 21-28 7 14

ppIgG

(2x2 gr/day)

Infected Infected+ppIgGInfected + Zink

1. 2. 3.

Inoc

ulat

ions

10 2

N = 6 N = 6 N = 6 (5)

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a: p<0.03; IgG vs. Zincb: p<0.03; IgG vs. Ctrl

c: p<0.02; IgG vs. Zincd: p<0.01; IgG vs. Ctrle: p<0.02; Zinc vs. Ctrl


Pre-clinical trial (Feacal bacteria)

Litter mix Weaning

Day 21-28 7 14

ppIgG

(2x2 gr/day)

Infected Infected+ppIgGInfected + Zink

1. 2. 3.

Inoc

ulat

ions

10 2

N = 6 N = 6 N = 6 (5)

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a: p<0.02; IgG vs. Zinc


The impact of dietary swine plasma immunoglobulins on intestinal microbiota and general health in weaner piglets

Poster:

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Safety: Infectability

On-column virus deactivation

Deactivation

Env. DNA Non-env. DNA

Env. RNA Non-env. RNA

Post production virus deactivation

Model viruses

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Removal

StabilityActivity

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Safety: Infectability (work in progess)

On-column deactivation with Na-caprylate

Infectability

Env. DNA Non-env. DNA

Env. RNA Non-env. RNA

Porcine Parvovirus (PPV)

Sample Infectability lvl

Neg. Ctrl -Pos. Ctrl (low PPV lvl) +

Pos. Ctrl (med PPV lvl) ++

Pos. Ctrl (high PPV lvl) +++IgG+PPV +++

IgG+PPV+caprylate pH 4.5 -IgG+PPV+caprylate pH 5.1 -

Stability

Indirect ELISA(E. coli)

0.00010.0010.010.11100

1

2

3

ppIgGPlasmano antigenEluate AEluate BEluate CEluate DEluate EEluate F

mg/ml

OD

450-

650

Activity

Removal

Fraction Titre Recovery (%)1

Fibrinogen precipitate 6.85 x 106 71.8Supernatant 3.49 x 104 0.3666Run through 2.28 x 104 0.2391

1st Wash 2.78 x 103 0.02922nd Wash (caprylate) 1.06 x 102 0.0011

3rd Wash (50°C) 3.07 x 102 0.0032Eluate (NaOH) 3.04 x 102 0.0032Regeneration 1.37 x 102 0.0014

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9.52 x 106

1Adjusted for sample volume

4-log reduction

Caprylate deactivates PPV


Summary• Swine production consume large amounts of antimicrobials (e.g. antibiotics

and dietary zinc oxide) especially for treating postweaning diarrhoea that– pollute the environment – develop microbial antibiotic resistance

• Natural porcine plasma immunoglobulins harbour anti-E. coli activity that– in vivo can reduce induced diarrhoea in weaner piglets– by reducing faecal haemolytic bacteria (incl. E. coli)– do not alter the intestinal microbiota in healthy weaner piglets

• Natural porcine plasma immunoglobulins exerts similar effects as dietary zinc oxide on reducing diarrhoeagenic E. coli in weaner piglets

• These results indicate that purified porcine immunoglobulins is a sustainable alternative to antibiotics and zinc

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Acknowledgements

Allan Lihme

Marie Bendix Hansen

Bodil Kjær Lindved

Kenneth Harlow

Michael Pålsson

VET(Knowledge Centre for Agriculture and Pig Research)

Sponsor:

Poul Bækbo

Claus Hansen

Chromatographies A/S

FoulumDANISH CENTRE FOR FOOD AND AGRICULTURE

Charlotte Lauridsen

Inger Marie Jepsen

& staff

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Peter M. H. Heegaard

Henriette T. Vorsholt

Heidi G. Andersen

Sophia Rasmussen

Mikael Lenz Strube

Mette Boye

Tim Kaare Jensen

Annie Ravn

Hans Skaaning

Maja Rosendahl

Nanna B. Jensen

Lars E. Larsen

Lise K. Kvisgaard

Hue 3xT

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