I POSSIBILI APPROCCI ALLA VALUTAZIONE DEL ... - Istituti

La risposta immunitaria a stressori infettivi e noninfettivi
nel suino: distinzione tra la risposta
costitutiva in interferon-alfa rispetto a quella virusindotta
Massimo Amadori, Elisabetta Razzuoli
IZSLER - Brescia
Background
Established view
A new conceptual
framework
Open problems
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Introduzione: l’allevamento del suino
Allevamento suino e uso del farmaco veterinario
Punti critici del ciclo zootecnico: lo svezzamento
Conseguenze fisio-patologiche sugli animali
Possibili strategie per un aumentato benessere e
riduzione dell’uso del farmaco
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L’allevamento del suino in Italia
Suino pesante: 10 milioni capi macellati / anno
Circuiti esterni: 3 milioni capi macellati / anno
Scrofe: 800.000 circa
Suini prodotti: 18 milioni nel complesso
Mancano all’ “appello” 5 milioni di animali !!
Di questi è interessante la scomposizione
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Gli aspetti preoccupanti
(Candotti P., Rota Nodari S., 2006)
Categorie: morti, “scarti” o “declassati”. Quindi:
1,8 milioni morti durante l’allattamento
0,25 milioni di scrofe e verri macellati
0,72 milioni morti entro 80 giorni di vita
0,54 milioni morti nel restante periodo
Rapporto ISTAT 2005: segnalati 800.000 lattoni
e 1.200.000 magroni macellati
2 milioni di suini macellati come “scarti” (11%)
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Motivi di tali perdite ?
Impianti tecnologici avanzati
Riduzione ore lavoro / capo
Strutture “hard”, alta competizione per risorse
e zone riposo
Qualità aria e pavimentazioni
Fasi giovanili: patologie sistemiche (PMWS)
Accrescimento: forme respiratorie
Scrofe: rimonta al 35%, sindromi mammarie,
osteo-articolari
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Uso degli antibiotici
Condizioni attuali: uso di mangimi medicati su
larga scala
Uso profilattico: inaccettabile
Farmaci iniettabili long acting : soggetti < 40
Kg
Soggetti malati: comunque numerosi
Il trattamento di questi è obbligatorio (D.Lvo
146/2001)
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Resistenza agli antibiotici
Parere Scientifico Congiunto 2009 di: ECDC,
EFSA, EMEA, SCENIHR
Antibiotico-resistenza di agenti di zoonosi:
in aumento in tutto il mondo
E’ necessario rafforzare la sorveglianza
Sviluppare nuovi antibiotici
Sviluppare nuove strategie di controllo
della antibiotico-resistenza
(http://www.efsa.europa.eu/cs/BlobServer/Scientific_O
pinion/1372.pdf?ssbinary=true )
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La situazione attuale
Preoccupanti i livelli di resistenza osservati con
Salmonella e Campylobacter spp
Possibile contributo di biocidi (disinfettanti,
antisettici e conservanti)
Livelli diversi nei Paesi UE
Difficile adozione di strategia unitaria
Fonti principali di contagio alimentare: macello
e processazione di alimenti
Precauzioni per fluorochinoloni e cefalosporine
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Ceppi Salmonella resistenti a Chinolonici
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Resistenze agli antibiotici e sanità pubblica:
un’incognita?
The New England Journal of Medicine
342: 1242-1249, 2000
Ceftriaxone-resistent salmonella infection
acquired by a child from cattle
Paul D. Fey et al.
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Fabbisogno di antibiotici: fronti di
intervento
Igiene zootecnica e benessere animale
Tempestività degli accertamenti diagnostici
Genetica animale e adattamento ambientale
(“forbice” da controllare)
Funzionalità del sistema immunitario innato ed
adattativo
Immuno-modulazione mirata in fasi critiche
dei cicli zootecnici
Quali basi concettuali ?
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stimoli
psicosensoriali
SISTEMA NERVOSO CENTRALE
sistema neuroendocrino
SISTEMA IMMUNITARIO
stimoli
antigenici
risposte
comportamentali
risposta
immunitaria
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The stress response
Immune response, stress and inflammation:
ancestral set of responses neutralization of
noxae perturbing body homeostasis
Cytokines : homeostatic agents
Effector mechanisms: similar in infectious and
non-infectious stress
For instance: IL-1 HPA + cerebral NA after
infection, electric shock or restraint
Brain IFN-α after infectious and noninfectious
stimuli
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La risposta immunitaria
Può essere rivolta verso:
STRESSORI INFETTIVI
STRESSORI NON INFETTIVI
Degenerazione, stress e distruzione tissutale
da cause varie (es. : alterazioni e disfunzioni
metaboliche, stress ox, lesioni articolari,
disturbi vascolari, ecc.)
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SISTEMA IMMUNITARIO
Sistema deputato al riconoscimento delle componenti
estranee all’organismo animale
Componenti della “clearance “ degli agenti di infezione
Fase Caratteristiche Meccanismo
Immediata (96 ore)
Non specifica
Inducibile
Non da memoria
Specifica, inducibile, dà
memoria, Comporta
linfociti specifici
Interferon, cellule NK
attivate da IFN
Linfociti T Citotossici,
interferon, anticorpi
specifici
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Attivazione del Sistema Immunitario Innato
1)PAMPs: strutture microbiche tipiche (LPS)
Riconoscimento mediato da: PRP (es. TLR),
RIG-like helicases (RIG-1 e MDA-5 per ac.
Nucleici virali), NLRs (es. per PG batterico)
2) DAMPS: da cellule danneggiate (es. acido
urico) segnalazione del “pericolo”
Quale teoria alla base del modello ?
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An innate sense of danger
(Matzinger P., 2002, Ann. N.Y. Acad. Sci. 961, 341-342)
Il paradigma “self – not self” è insufficiente
Presenza fisiologica di cellule T e B autoreattive
ma innocue, proteine del latte, Ag
alimentari, batteri commensali, feti, sperma:
fenomeni non spiegati dal paradigma
Driving force: riconoscimento del “pericolo”
Danger: stress e/o distruzione tissutale:
rilascio di segnali (alarmine o DAMPs)
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Il ruolo delle “alarmine”
DAMPs o “alarmine”: prodotto di cellule
necrotiche, stressate e degenerate
Alarmine: HMGB1 (e istoni in generale): lega
TLR2 e 4, RAGE
ROS ciclofillina A, proteine S100 (A8, A9):
legame a TLR-4
Trasduzione di potenti segnali in senso proflogistico:
SAPK cJun NH2, p38 MAPK
HSP72: antagonista naturale di tali risposte
(vedi bovine da latte !)
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Alarmine: secrezione di istoni in cellule BHK-21
infettate da FMDV O1 Losanna
1 2 3 4 5 6 7 8
30’ k30 120’ k120’ 240’ k240’ 360 k360’
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Infiammazione mortale, no infezione.
Il mistero SIRS
(Systemic Inflammatory Response Syndrome)
Esempio: pazienti con gamba rotta, dopo 2 giorni
febbre altissima, shock, resp. artificiale
Ipotesi trad. Ridotto flusso ematico all’intestino,
aumento permeab. Intestinale, batteriemia
In realtà: quadro plasmatico sterile in vena porta
Plasma: alto contenuto di DNA, MITOCONDRI !
Per SII: mitocondri = Batteri = risposta flogistica
HBS (Hemorragic Bowel Syndrome) nelle bovine da
latte ??
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Organizzazione della risposta flogistica:
il ruolo dell’inflammosoma
Indotto da segnali di PAMPs e DAMPs
Complesso di oligomeri NLRs/adattatore (ASC)
caspasi 1 IL-1β
3 classi: Nalp1, Nalp3, IPAF (diverso effettore)
IL-1β MyD88: trasduzione di segnale come
PAMP !!
Attivazione: variazioni ioniche (K+), ATP, acido
urico, stress ox
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Ruolo degli inflammosomi
Discriminare l’ambiente microbico e non
microbico per individuare le condizioni di
“pericolo” (Danger Theory)
Repertano segnali infiammatori endogeni in
assenza di infezioni microbiche (vedi SIRS!)
Sono alla base dell’efficacia di alcuni adiuvanti
come Al (OH) 3 DC inflammosoma
NALP3 IL-1β
Collegamento immunità innata / adattativa
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Come si manifesta la risposta a
stressor non-infettivi ?
1) Fenomeno flogistico citochine e chemochine
circuiti regolazione omeostatica
2) Risposta linfocitaria allo stress (specie linf. T γδ):
Riconoscimento di “neo-antigeni” e antigeni da stress:
T10, T22, CD1c (DC!), IPP, ATPseF1, altri Ag MHC-like
(MICA, MICB, H60). Ovvero:
Danno epit. Linf.Tγδ DC risposta T conv.
(inversione del flusso consueto)
Lymphoid Stress-Surveillance Response
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Una nuova frontiera: il concetto di
“Behavioral Immunization”
Precedenti blandi stress sono “educativi”
Fenomeni immunitari implicati ?
Topi deficitari di cellule T fronteggiano peggio lo
stress mentale
Stress: aumentato traffico di linfociti al cervello
Effetto protettivo verso nuovo stress
Minore ansietà dopo vaccinazione con peptide
SNC
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Esistono evidenze in medicina veterinaria?
Lymphoid Stress-Surveillance Response: modelli
umani e murini
Secrezione di citochine in risposta a stressors
non-infettivi: evidenziata anche in bovini e suini
Interferon I (specie IFN-α) gioca un ruolo
regolatorio centrale
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Interferon: a dogma in immunology
Dogmas / paradigms = serendipitous
Discovery of new components of the immune
system
Interferon = no exception to this rule
1957 = Isaacs and Lindenmann discovery of
a potent antiviral substance accumulated in
embryonated eggs
50 years later: most immunologists share the
same view: antiviral activity + “accessory
properties”
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IFNs as homeostatic agents
Three types of IFNs (I, II and III)
Type I IFN: 10 sub-families (α, β, ω, τ, etc.); many α subtypes
IFN-α : a highly diversified system (13 genes and 5 pseudogenes
in humans; at least 14 genes and 2 pseudogenes in pigs) should
serve diverse functions and effector mechanisms
Low levels of IFN α/β in tissues of healthy subjects:
constitutive expression
Steady-state role under health conditions
Most important: frequent involvment in the stress
response
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The physiological IFN response
IFN is produced under health conditions: e.g.
after meals and drinking
Not obligatorily related to infection status
This is true of the whole type I family
IFN-β : maintenance of memory T cells
IFN-τ : survival of the conceptus in ruminants
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Aims of the physiological IFN response
Strong cellular responses to viruses and
cytokines
To control the effects of growth factors
To prevent unnecessary harmful inflammatory
responses in tissues
Default function of IFN-α
It would complement other established
biological activities after infectious and noninfectious
IFN-inducing stimuli
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In vitro activity of IFN-α
Very low concentrations (0.05 – 5 U/ml)
down-regulate CD14 expression in swine
PBMC and PAM, as opposed to higher
concentrations
Signalling by LPS/LPS binding protein is
inhibited
Released CD14: potent scavenging system for
LPS
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How does the control action take place?
A direct modulation of the inflammatory
response in target cells
Release of second messengers for
neighbouring cells
Neighbouring cells would be thus instructed to
down-regulate the inflammatory response
An in vivo propagation of these signals to
distant sites would be possible as well.
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The IFN-α response in farm animals
Low-titered IFN-α response in pigs after early
weaning and transportation (calves, too)
Early weaning: activation of inflammatory
cytokine genes in the small intestine and upregulation
of IFN-γ in PBMC
IFN-α at weaning: (1) A SPF pig model . (2) A
conventional pig model
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The SPF pig study
1 litter of L x Lw SPF piglets (n. 8)
Matched for weight at day 20 of age (4.9±0.9 versus
5.4±0.8 Kg in treatment and control groups, respectively)
and weaned one day later.
At weaning, 4 piglets: 20 IU freeze-dried human IFNalpha
/ Kg b.w. for 10 days in RG lactose per os. 4 piglets:
placebo (the same mass of RG lactose) in a separate cage.
Daily clinical inspections. Blood samples at days –1 / +3 /
+21 with respect to weaning
Individual weight controls at days –1/ +21 / +30
RT real time PCR for inflammatory cytokine genes in
PBMC. Interferon-α and TNF-α bio-assays on serum
samples (MDBK and WEHI 164 cells).
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Results obtained on SPF piglets
No clinical difference between treated and control animals
Daily weight gain greater in IFN alpha-treated groups between days -1
and +21 and also between days 21 and 30; the effect of the treatment between
days –1 and + 30 was almost significant (p

log2(n-fold)
N-fold gene expression values by the 2 -ΔΔCt method
(basis: mean value at day -1)
Housekeeping gene: TATA box-binding protein gene (TBP)
2.0
1.0
0.0
-1.0
-2.0
-3.0
-4.0
-5.0
-hIFN-alpha
IFNA
+hIFN-alpha
-hIFN-alpha
+hIFN-alpha
-hIFN-alpha
IFNG IL1B IL6
TNF
+hIFN-alpha
α
-hIFN-alpha
β
+hIFN-alpha
b
-hIFN-alpha
a
+hIFN-alpha
3d 21d 3d 21d 3d 21d 3d 21d 3d 21d
-hIFN-alpha
+hIFN-alpha
-hIFN-alpha
+hIFN-alpha
-hIFN-alpha
+hIFN-alpha
-hIFN-alpha
+hIFN-alpha
-hIFN-alpha
+hIFN-alpha
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Aims:
The conventional pig model
1) To check the oral, IFN-α treatment under
practical, large-scale conditions (in drinking
water)
2) To evaluate the endogenous IFN-α
response
3) To assess the influence of the weaning age
4) To characterize such a stress-induced
response
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The field trial: role of the endogenous IFN-α response
39, LxLW piglets, of a healthy, farrow-to-finish
herd, randomly allocated to:
Group 1: weaned at 28 days (19 pigs, 2 litters)
Group 2: weaned at 22 days (20 pigs, 2 litters)
Group 3: weaned at 22 days and orally treated
with hIFN-α in drinking water (1 IU / Kg
b.w. over 10 days) (20 piglets, 2 litters).
Clinical checks and blood samplings at days -1 /
+6 / +12 with respect to weaning
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Clinical findings
All piglets remained healthy during the trial
No clinical difference between early and late weaning
groups. DMWG: greater in the late weaning group
(28 days) and in IFN-treated animals (both: +1.5
Kg on average at 48 days of age) (p

The IFN-α response in serum samples
Interferon alpha-positive
alpha positive pigs out of the total
Groups Day -1 Day + 6 Day + 12
1 (weaned at 28 days) 1/19 7/19* 0/19
2 (weaned at 22 days) 1/20 20/20 1/20
3 (weaned at 22 days
and IFN alphatreated)
0/20 18/20 0/20
*Significantly different, p

Gel-filtration
Gel filtration on a Sephadex G-75 75 column of IFN-α IFN in samples
from group 2 pigs at day + 6
Kav
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
Fractions checked by a bioassay
on MDBK cells
Serum
* Also shown by rec. IFN-α 1
0
y = -0,5959x+3,1898
27-30 27 30 kDa*
17-18 17 18 kDa*
< 14 kDa
(Apparent MW)
R 2 = 0,9909
4,1 4,2 4,3 4,4 4,5 4,6 4,7 4,8 4,9
PM log
Log MW
Fractions checked by ELISA
with mAbs F17 and K9
PBMC NP-40 lysates
Also: 58 kDa (dimer)
and 41 kDa
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Flow Cytometry: Cytometry:
analysis of intracellular IFN-α in
fixed and permeabilized swine PBMC at day +6
IFN-α positive samples out of the total:
• Group 1: 4 / 19 (0.6 – 3.9% positive cells)
• Group 2: 10 / 20 (0.6 – 8.8% positive cells)
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Control
Anti-IFN α mAb
Flow cytometry assay for intracellular porcine IFN-α
in fixed and permeabilized swine PBMC at day +6
2% IFN α−positive cells on average
(0.1 – 0.3% in 2-3 month-old pigs)
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RT real time PCR for IFN-α IFN genes (group group 2 pigs) pigs
Day -1
Campione IFN-α 1 IFN-α 2 IFN-α 3 IFN-α 4 IFN-α 5/6 IFN-α 7/11 IFN-α 8 IFN-α12
25T0 - - - - + - - +
26T0 - - - - + - - +
28T0 - - - - - - - +
29T0 - - - - + - - +
30T0 - - - - + + - +
31T0 + - - - + - - -
32T0 + + - - - + - -
33T0 - - - - + - - -
34T0 + + - - + + - +
Day +6: no expression evidenced
Day +12
Campione IFN-α 1 IFN-α 2 IFN-α 3 IFN-α 4 IFN-α 5/6 IFN-α 7/11 IFN-α 8 IFN-α12
25T+12 + + - - - - - -
26T+12 + + - - - - - -
28T+12 + + - - + - - +
29T+12 - - - + - - -
30T+12 - - - - + - - -
31T+12 - + - - + - - +
32T+12 + + - - - - - -
33T+12 - - - - + - - -
34T+12 + + - - + + - +
Assays carried out according to Cheng G. et al., (2006), Gene 382, 28-38
(No valid results for IFN α 10 gene)
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IFN-α gene expression at days -1, +6, +12
Day -1
Day +12
Day +6
Day +12
Day +12
Day -1
Day +6
Day -1
RT real time PCR for IFN-α 5/6
The test was carried out on
PBMC of the same pig at days –1
/ +6 / +12 with respect to weaning
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Constitutive and virus-induced expression
Previous data on PK-15 cells in vitro:
Virus-induced expression: IFN-α 2/3/4/8/9/10/13
Constitutive expression: IFN-α 1/2/5/6/7/11/12
Truncated Porcine IFN-α genes: IFN-α 1/2/3/11
Genes with N-glycosylation site: IFN-α 7/9/10/11
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Issues raised by the field trial and in vitro tests
Is this pattern of response also applied to
infectious (viral) stressors ?
Are inflammatory cytokines released at
weaning implied in the modulation of type I
IFN system ?
This is the reason why we investigated IFN
genes and proteins in a relevant model of in
vitro cultured swine PBMC from healthy, 70 to
80-day old animals, far away from the
weaning stress.
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In vitro tests
IFN proteins (α nd γ) and IFN-α gene expression
in PBMC cultures of 70/80-day old pigs
Time 0
Unstimulated control
Priming (100 IU/ml IFN-a)
Priming + Paramixovirus (NDV) stimulation
NDV, only
Supernatant collection / RNA extraction after 18
hours of culture
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In vitro results
IFN-α1, α2, α4, α5/6 and α7/11 genes were shown to be
involved in constitutive expression in uncultured PBMC
(Time 0 samples).
Further IFN-α genes were then expressed in culture.
Interestingly, expression was often shown in unstimulated
cultures, as well.
As a result, NDV stimulation often caused only increased
expression compared with unstimulated cultures
Alpha 9 expression: virus-dependent, associated to
protein release
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Un ruolo di IL-1β ?
IL-1β suina ricombinante può indurre in vitro
tracce di IFN-α.
Colture di PBMC e di sangue in toto
Per quanto concerne i geni IFN-α, IL-1β si
comporta come NDV, specie per quanto
concerne il gene α9
La co-presenza di TNF-a modula
l’espressione dei geni IFN-α da parte di IL-
1β
Ruolo di inflammosoma in vivo ?
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A possible explanation of the IFN-α response ?
Weaning
(alarmins)
Intestinal cell damage NLRs
inflammosomes IL-1β
Type I IFN
(Control action)
IL-18
IL-12
Ads. LPS
(Amplification of
antibacterial
immune
response)
(α / β) (control action ?)
IFN-γ: danger ! (Imbalance Th17/Treg in SI?)
Up-regulation of crypt cell response to LPS, downregulation
of repair processes in ECM, increased intestinal
permeability: post-weaning diarrhoea !!
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Response to LPS of intestinal cells
Porcine intestinal epithelial cells: expression
of TLR-4 / MD2 complex (Moue et al., 2008)
Fully competent for a response to LPS (IL-8
for example)
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Which circumstantial evidence ?
IL-12 + IL-18 i.p. diarrhoea in mice
Histologically, lesions in SI similar to those of pigs after
weaning. Effects abrogated in IFN-γ KO mice (Chikano
S. et al., 2000, Gut, 47, 779-786)
IFN-γ increases intestinal epithelial permeability
(Beaurepaire C. et al., 2009)
In piglets, IL-1β gene is up-regulated in most parts of
the intestine after weaning (Pié S. et al., 2004)
In piglets, plasma IL-1β is increased after weaning
(McCracken B. et al. 1995)
In vitro, porcine IL-1β induces IFN-α in leukocyte
cultures.
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Conclusions: (1)
An oral, low-dose IFN-α treatment was shown to
modulate the stress of early weaning
No clinical difference, but DMWG was greater
IFN-γ is the main target of the control action in PBMC
This is justified by precise biological properties of IFN-γ
IFN-α: possible amplification loop of the IL-1β response
TNF-α and IL-6 play instead a minor role in the 1st week
after weaning
A tendency to a control action in the 3rd week after
weaning (correlation with WG data in the field trial !)
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Conclusions: (2)
Endogenous IFN-α is involved in the pigs’
adaptation strategy to weaning
The requirement is probably lesser in 4 -, compared
with 3-week old piglets.
A transcriptional feed-back control on IFN-α is
exerted at day 6 after weaning.
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Take home message
Type I IFNs can act as important homeostatic
agents in the response to environmental stressors
The immune system displays similar responses to
both infectious and non-infectious stressors
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Use of IFN-α in veterinary medicine
Field trials as immunomodulator: IFN
administration and induction of endogenous IFN
Environment is critical: poor efficacy in the
presence of overwhelming infectious pressure
Protocols to be adopted in “problem” herds
and/or in farms with abnormal values of clinical
immunological parameters.
Evidence of reduced immunological competence
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IFN-α trial 1: anamnestic data
In a farrow-to-finish farm with 520 sows, a respiratory form was
observed in 40-day old piglets (20 days after weaning) :
» High morbility: 60%
» Dry cough
» Mortality: 7-12%
» Low-grade anorexia
15% of piglets experienced progressive wasting
Virological tests on affected animals revealed both PRRSV (Eu and
USA) and PCV2
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Protocol
Trial scheme
Oral administration of freeze-dried IFN-α for 15 days after weaning
Product constantly fed (10 IU/Kg bw) by an automatic device through the
drinkers
A more strict control of good farming practices
Controls: initial and final weights of pigs, number of dead and waste pigs,
mean daily weight gain
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Group Pigs
Mean
weight
at 22 days
(Kg)
Results
Mean weight
at 86 days
(Kg)
Daily weight
gain (Kg)
Dead and
waste pigs
(%)
IFN-α treated 280 5.6 37.1 0.48 1.4*
Controls 280 5.6 33.8 0.44 4.9*
*P

IFN-α trial 2
(Candotti P., Rota Nodari S., 2003)
Aim: to evaluate the
clinical efficacy of oral
IFN-α treatment against
a reproductive
syndrome ( abortion,
agalaxia and
periparturient mortality)
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Use of oral IFN-α in an outbreak of reproductive PRRS
In july 2002 a herd of 400 sows was affected
by a typical PRRS outbreak, characterized:
» Early parturition
» Agalaxia
» High mortality of piglets
Oral administration in the farrowing cage,
starting at day 7 before the alleged parturition
date till day 3 after farrowing
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100%
80%
60%
40%
20%
0%
Results: sows
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Healthy
Diseased
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100
80
60
40
20
0
Week 1
Week 2
Week 3
Week 4
Results: piglets
Week 5
Week 6
Week 7
Week 8
Week 9
Weaned %
Mortality %
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IFN-α trial 3: reproductive PRRS
(Candotti P., Rota Nodari S., 2003)
In January 2002 a herd of 1100 sows was
affected by reproductive PRRS:
» Enzootic abortion
Oral administration of IFN-α was carried out
on all pregnant sows in the ad libitum food
ration for 15 days in a row
The treatment was repeated 15 days later
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70
60
50
40
30
20
10
0
June
August
October
Results
December
Abortions
February
April
June
August
October
December
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Dose/response curve of IFN-α
Foundation in the dose/response curve
Immune effector functions: bell-shaped curve
Positive/decrease/even reversion (e.g. Ab response
to sheep RBC in mice)
Concept: low dose priming / high dose suppression
Timing and concentration: crucial roles (subtypes?)
At odds with antiviral / anti-proliferative activities:
it increases with increasing IFN-α concentrations
IZSLER Brescia

Homeostatic role of IFN-α:
truck transportation of calves
Long-distance journeys of calves:
Serious sensory dullness
Dehydration (HCT, serum protein, albumin, Hb)
Adrenal response (glucose, urea)
PO 4 increase e Fe decrease
Detection of IFN-α in serum
IZSLER Brescia

Risposta al trasporto: citochine e risposta APP+
concentrazione
40
35
30
25
20
15
10
5
0
in vitelli trasportati dalla Polonia
T-2 T-1 T0 T+4 T+15
tempo
IL-6 (ng/ml)
TNF alfa (ng/ml)
Aptoglobina (mg/dl)
Elevata risposta in IL-6 e TNF-alfa (carattere compensativo) prima e dopo il trasporto
Risposta APP+
Elevata pressione infettante in allevamento di origine
IZSLER Brescia

IFN-alfa IFN alfa e risposta adattativa in vitelli trasportati
7
6
5
4
3
2
1
0
dalla Polonia
T-2 T-1 T0 T+4 T+15
Tempo
IFN-alfa
Negativi
Positivi
Risposta in IFN-alfa in tutti gli animali nel centro di raccolta e dopo il trasporto
Diminuzione a 4 giorni dall’arrivo in Italia
Conferma del suo ruolo adattativo di tale citochina
picco di citochine infiammatorie risposta IFN-alfa
IZSLER Brescia

La filiera ANAFI:
il trasporto dei bovini di breve durata
5 trasporti controllati di torelli Frisoni di 6-12
mesi al Centro Genetico ANAFI di Cremona
(totale 26)
Dicembre 2008, Gennaio 2009, Aprile 2009,
Giugno 2009, Luglio 2009
200 – 300 Km, 3-6 ore*, Torino-Cuneo/Cremona
Sopralluoghi a T-4 (esame clinico ed etologico),
T0 (carico-scarico), T+4, T+15, T+30
Prelievi ematici nelle stesse date
Riprese filmate da T-4 a T+4
IZSLER Brescia

E’ importante la componente climatica nei
trasporti animali ? Suddivisione dei trasporti di
breve durata su base stagionale
Si possono distinguere:
2 trasporti invernali (range: 5,7-13,7 °C; 2,6-
9,4 °C ). Trasporti 1 e 2
1 trasporto in stagione termo-neutrale (range
11,3-16,1 °C) Trasporto 3
2 trasporti estivi (range 23-33 °C, THI 46-
73,5). Trasporti 4-5. Temp. > superiori a
quelle prospettate nel doc. Commissione UE
IZSLER Brescia

Lesioni muscolari, enzima CK plasmatico
900
800
700
600
500
400
300
200
100
0
T-4 T0 T+4 T+15 T+30
CK
giorni dal trasporto
Andamento differente nel trasporto 1 a causa di forti interazioni
agoniste di 2 torelli, mai tenuti in gruppo prima. Elevata
prevalenza di risposte IFN-alfa sino a T+15 !!
Gruppo 1
Gruppo2
Gruppo3
Gruppo4
Gruppo 5
IZSLER Brescia

Risposta negativa (APP-) e positiva di fase acuta
45
40
35
30
25
20
15
10
5
0
Albumina
T-4 T0 T+4 T+15 T+30
giorni dal trasporto
La risposta APP+ nel gruppo 5 è accompagnata da risposta APP-
(ipo-albuminemia) a T+15, come evidenziato nelle bovine da latte
nel periparto. Forte correlazione con la risposta in IFN-alfa !!
Gruppo 1
Gruppo2
Gruppo3
Gruppo4
Gruppo 5
IZSLER Brescia

Citochina IL-6 e risposta di fase acuta
10000
1000
100
10
1
T-4 T0 T+4
giorni dal trasporto
T+15 T+30
Andamento atteso per i trasporti 2 e 5: come atteso, in assenza
di lesioni, RFA dipende da incremento ad alto titolo di IL-6
plasmatica.
IL 6
Gruppo 1
Gruppo2
Gruppo3
Gruppo4
Gruppo 5
IZSLER Brescia

Trasporto breve: conclusioni
Non è possibile considerare l’impatto del trasporto
avulso dal contesto ambientale (origine +
destinazione): strategia adattativa globale.
L’esito finale in termini di sanità e benessere animale
dipende dal complesso di condizioni pre e post
trasporto, oltre che dal trasporto stesso.
2 strategie di adattamento:
A basso impatto infiammatorio (gr. 3): incremento e/o
allungamento delle alterazioni da stress acuto
(cortisolo, NEFA, glucosio, lattato)
Ad alto impatto infiammatorio (gr. 1): da traumi nel
trasporto (CK, APP+, IFN-alfa, NEFA)
IZSLER Brescia

The “hinge” role of IFN-α
A “hinge” role of IFN-α between innate and
adaptive immunity is widely recognized
PDC IFN-α type and extent of the
adaptive immune response
The concept should now be widened
IFN-α would also promote a balance between
danger and response
A homeostatic control action: the “spring”
mechanism
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The “spring” mechanism of IFN-α
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Flow chart of actions
Start of innate immunity: IFN-α released at
moderate/high concentrations after TLR
recognition of microbes
Later on clearance of microbes decrease
of IFN-α release
Shift to an inflammatory control action based
on the transcriptional control of inflammatory
cytokine genes and PAMP receptors (CD14)
Major tissue damage is avoided
The system gets at a “stand-by” status
IZSLER Brescia

Tuning of the IFN-α response
Tuning of the response of paramount
importance
Detrimental effects because of high therapy
dosage, overexpression in transgenic mice,
genetic defects of regulation even without
infection
This confirms the induction of IFN-α under
health conditions and the need for a strict
control of this response
Excessive concentrations of IFN-α checked by
Ab responses in the host (self /not self ??)
IZSLER Brescia

A note of caution
Several features need to be investigated
4 issues:
which effector cell populations ?
how are they activated ?
how can they sense different
concentrations of IFN-α ?
how do they propagate the signal ?
As many issues for future research !!
IZSLER Brescia

Acknowledgements
Dr. Paolo Candotti, IZSLER, Brescia
Dr. Ivonne Laura Archetti, IZSLER, Brescia
Dr. Barbara Begni, IZSLER, Brescia
Antonio Cristiano
Cinzia Mantovani
Lab technicians, IZSLER
IZSLER Brescia

Thank you for
the attention !
IZSLER Brescia