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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
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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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Threat of microbial antibiotic resistance
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USA (CDC 2013):
Europe (ECDC/EMEA 2009):
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Sales of veterinary antimicrobial agents in 26 EU/EEA countries
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5th ESVAC report 2013
PCU = animalpopulation correction unit
11/01/201711 January, 2017From: WHO 2012
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Antimicrobial consumption in Denmark
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30% of total Danish antibiotics goes to PWD
treatment
Antimicrobial consumption in Denmark
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Dietary zinc oxide has a negative effect on the environment
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Alternatives to antibiotics are (also) needed in swine production
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• 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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Light chain
Heavy chain
Immunoglobulin
Purified Pig IgG = ppIgG
Natural antibodies against Postweaning dirrhoea
Hedegaard et al (2016) PLoS One
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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
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Reactivity against bacteria
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Indirect ELISA
= Bacteria lysate
TMB
= ppIgG
= HRP:anti-pig
IgG antibody
Hedegaard et al (2016) PLoS One
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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Shedding of ETEC (faeces)
Enterobacteriaceae in ileum
(p=0.0017)
(p=0.001)
Hedegaard et al (2016) PLoS One
PWD: experimental model
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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
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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
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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
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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