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La risposta immunitaria a stressori infettivi e non-infettivi nel suino: distinzione tra la risposta

costitutiva in interferon-alfa rispetto a quella virus-indotta

Massimo Amadori, Elisabetta RazzuoliIZSLER - Brescia

Background

Established view

A new conceptualframework

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.Lvo146/2001)

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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 controllodella antibiotico-resistenza(http://www.efsa.europa.eu/cs/BlobServer/Scientific_Opinion/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 infectionacquired 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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SISTEMA NERVOSO CENTRALE

SISTEMA IMMUNITARIO

sistema neuroendocrino

stimoli antigenici

stimoli psicosensoriali

rispostaimmunitaria

risposte comportamentali

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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 non-infectious stimuli

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La risposta immunitaria

Può essere rivolta verso:

STRESSORI INFETTIVI

STRESSORI NON INFETTIVI

Degenerazione, stress e distruzione tissutaleda 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

Fase Caratteristiche Meccanismo

Immediata (<4 ore)

Non specifica

Innata

Non dà memoria

Atività del SRE

Lisozima, complemento, anticorpi naturali, cellule NK

Precoce (4-96 ore)

Non specifica

Inducibile

Non da memoriaInterferon, cellule NK attivate da IFN

Tardiva(>96 ore)Specifica, inducibile, dàmemoria, Comporta linfociti specifici

Linfociti T Citotossici, interferon, anticorpi specifici

Componenti della “clearance “ degli agenti di infezione

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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 auto-reattive ma innocue, proteine del latte, Agalimentari, 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 pro-flogistico: 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

30’ k30 120’ k120’ 240’ k240’ 360 k360’

1 2 3 4 5 6 7 8

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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 adiuvanticome Al (OH)3 DC inflammosomaNALP3 IL-1β

Collegamento immunità innata / adattativa

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Come si manifesta la risposta astressor non-infettivi ?

1) Fenomeno flogistico citochine e chemochinecircuiti 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 peptideSNC

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Esistono evidenze in medicina veterinaria?

Lymphoid Stress-Surveillance Response: modelli umani e murini

Secrezione di citochine in risposta a stressorsnon-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 + “accessoryproperties”

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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 pseudogenesin humans; at least 14 genes and 2 pseudogenes in pigs) shouldserve 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 inflammatoryresponses in tissues

Default function of IFN-α

It would complement other establishedbiological activities after infectious and non-infectious 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 swinePBMC and PAM, as opposed to higherconcentrations

Signalling by LPS/LPS binding protein isinhibited

Released CD14: potent scavenging system forLPS

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How does the control action take place?

A direct modulation of the inflammatoryresponse in target cells

Release of second messengers forneighbouring cells

Neighbouring cells would be thus instructed todown-regulate the inflammatory response

An in vivo propagation of these signals todistant 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 earlyweaning and transportation (calves, too)

Early weaning: activation of inflammatorycytokine genes in the small intestine and up-regulation of IFN-γ in PBMC

IFN-α at weaning: (1) A SPF pig model . (2) Aconventional 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 versus5.4±0.8 Kg in treatment and control groups, respectively) and weaned one day later.

At weaning, 4 piglets: 20 IU freeze-dried human IFN-alpha / 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 serumsamples (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 betweendays –1 and + 30 was almost significant (p<0.06).

Evidence of a low-titered serum IFN-alpha response in control animals, only

No evidence of serum TNF-α response but in 1 control pig at day +21

In RT real time PCR, IFN-γ was significantly up-regulated at day +3; expression significantly lower at days +3 and +21 in treated piglets.

TNF-α expression significantly different between sampling times.

IL-1β significantly increased in IFN-treated pigs at days +3 and +21

IL-6 at day +3, only.

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N-fold gene expression values by the 2-ΔΔCt method (basis: mean value at day -1)

Housekeeping gene: TATA box-binding protein gene (TBP)

IFNA

b

a

IL6

α

β

IFNG IL1B TNF

-5.0

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0 -h

IFN

-alp

ha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

-hIF

N-a

lpha

+hI

FN-a

lpha

3d 21d 3d 21d 3d 21d 3d 21d 3d 21d

log2

(n-fo

ld)

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The conventional pig model

Aims:

1) To check the oral, IFN-α treatment under practical, large-scale conditions (in drinkingwater)

2) To evaluate the endogenous IFN-αresponse

3) To assess the influence of the weaning age

4) To characterize such a stress-inducedresponse

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The field trial: role of the endogenous IFN-α response

39, LxLW piglets, of a healthy, farrow-to-finishherd, 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 treatedwith 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 weaninggroups. 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<0,001)

Correlated to lower serum TNF-α levels in the 3rd week after weaning

Blood samples were investigated for viral infections: all samples tested negative for Influenza A, PRRS, PCV-2, Pseudorabies viruses in PCR and Abassays

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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 alpha-treated)

0/20 18/20 0/20

*Significantly different, p<0.01

InterferonInterferon alphaalpha--positivepositive pigspigs out of the totalout of the total

The IFN-α response in serum samples

Assay: VSV cpe inhibition assay on MDBK cells, calibrated with Porcine rec. IFN-α1

Low titres: 1-27 IU/ml

Activity neutralized by anti-Porcine IFN-α1 mAb

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GelGel--filtrationfiltration on a on a SephadexSephadex GG--75 column of IFN75 column of IFN--αα in in samplessamplesfromfrom groupgroup 2 2 pigspigs at day + 6at day + 6

2727--30 30 kDa*kDa*

1717--18 18 kDa*kDa*

< 14 < 14 kDakDa

R2 = 0,9909

00,10,20,30,40,50,60,70,8

4,1 4,2 4,3 4,4 4,5 4,6 4,7 4,8 4,9

PM log

Kav

y = -0,5959x+3,1898

SerumSerum

Fractions checked by a bioassayon MDBK cells

Log MW

PBMC NP-40 lysates

Also: 58 kDa (dimer)

and 41 kDa

Fractions checked by ELISA with mAbs F17 and K9

* Also shown by rec. IFN-α1

(Apparent MW)

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FlowFlow CytometryCytometry: : 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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Anti-IFN α mAbControl

Flow cytometry assay for intracellular porcine IFN-αin 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 RT realreal time PCR time PCR forfor IFNIFN--αα genesgenes ((groupgroup 2 2 pigspigs))Day Day --11

Day +12Day +12

Day +6: no Day +6: no expressionexpression evidencedevidenced

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 + + - - + + - +

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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Day -1

Day +6

Day +6

Day -1

Day +12

Day +12

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

IFN-α gene expression at days -1, +6, +12

Day +12

Day -1

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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 expressionin 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 unstimulatedcultures, as well.

As a result, NDV stimulation often caused only increased expression compared with unstimulated cultures

Alpha 9 expression: virus-dependent, associated toprotein release

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Un ruolo di IL-1β ?

IL-1β suina ricombinante può indurre in vitrotracce 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 Intestinal cell damage NLRs(alarmins)

inflammosomes IL-1β

Type I IFN

(α / β) (control action ?)

IFN-γ: danger ! (Imbalance Th17/Treg in SI?)

Up-regulation of crypt cell response to LPS, down-regulation of repair processes in ECM, increased intestinalpermeability: post-weaning diarrhoea !!

(Control action)

(Amplification of antibacterial

immune response)

IL-18 IL-12

Ads. LPS

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Response to LPS of intestinal cells

Porcine intestinal epithelial cells: expressionof 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 (ChikanoS. 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 leukocytecultures.

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Conclusions: (1)

An oral, low-dose IFN-α treatment was shown tomodulate 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 weekafter 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 -, comparedwith 3-week old piglets.

A transcriptional feed-back control on IFN-α isexerted at day 6 after weaning.

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Take home message

Type I IFNs can act as important homeostaticagents in the response to environmental stressors

The immune system displays similar responses toboth 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” herdsand/or in farms with abnormal values of clinicalimmunological 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 wasobserved 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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Trial scheme

Protocol

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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Results

Group Pigs

Mean weight

at 22 days(Kg)

Mean weightat 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<0,005 (Fischer test)

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IFN-α trial 2(Candotti P., Rota Nodari S., 2003)

Aim: to evaluate the clinical efficacy of oralIFN-α treatment againsta reproductivesyndrome ( 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 affectedby 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 parturitiondate till day 3 after farrowing

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Results: sows

0%

20%

40%

60%

80%

100%

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9

Healthy

Diseased

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Results: piglets

0

20

40

60

80

100W

eek

1

Wee

k 2

Wee

k 3

Wee

k 4

Wee

k 5

Wee

k 6

Wee

k 7

Wee

k 8

Wee

k 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 wasaffected 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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Results

010203040506070

June

Augu

stO

ctobe

rDe

cem

ber

Febr

uary

April

June

Augu

stO

ctobe

rDe

cem

ber

Abortions

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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 responseto 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

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Long-distance journeys of calves:

Serious sensory dullness

Dehydration (HCT, serum protein, albumin, Hb)

Homeostatic role of IFN-α: truck transportation of calves

Adrenal response (glucose, urea)

PO4 increase e Fe decrease

Detection of IFN-α in serum

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05

10152025303540

T-2 T-1 T0 T+4 T+15

tempo

conc

entraz

ione

IL-6 (ng/ml)

TNF alfa (ng/ml)

Aptoglobina (mg/dl)

Risposta al trasporto: Risposta al trasporto: citochinecitochine e risposta APP+e risposta APP+

in vitelli trasportati dalla Poloniain vitelli trasportati dalla Polonia

Elevata risposta in IL-6 e TNF-alfa (carattere compensativo) prima e dopo il trasporto

Risposta APP+

Elevata pressione infettante in allevamento di origine

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01234567

T-2 T-1 T0 T+4 T+15

Tempo

IFN-alfa

Negativi

Positivi

IFNIFN--alfa e risposta alfa e risposta adattativaadattativa in vitelli trasportati in vitelli trasportati

dalla Polonia dalla Polonia

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

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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

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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 (range11,3-16,1 °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

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Lesioni muscolari, enzima CK plasmatico

CK

0

100

200

300

400

500

600

700

800

900

T-4 T0 T+4 T+15 T+30

giorni dal trasporto

Gruppo 1Gruppo2Gruppo3 Gruppo4Gruppo 5

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 !!

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Risposta negativa (APP-) e positiva di fase acutaAlbumina

0

5

10

15

20

25

30

35

40

45

T-4 T0 T+4 T+15 T+30

giorni dal trasporto

Gruppo 1Gruppo2Gruppo3 Gruppo4Gruppo 5

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 !!

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Citochina IL-6 e risposta di fase acuta

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

1

10

100

1000

10000

T-4 T0 T+4 T+15 T+30

giorni dal trasporto

Gruppo 1Gruppo2Gruppo3 Gruppo4Gruppo 5

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Trasporto breve: conclusioniNon 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)

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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 betweendanger 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 decreaseof IFN-α release

Shift to an inflammatory control action basedon the transcriptional control of inflammatorycytokine genes and PAMP receptors (CD14)

Major tissue damage is avoided

The system gets at a “stand-by” status

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Tuning of the IFN-α response

Tuning of the response of paramountimportance

Detrimental effects because of high therapydosage, overexpression in transgenic mice, genetic defects of regulation even withoutinfection

This confirms the induction of IFN-α under health conditions and the need for a strictcontrol of this response

Excessive concentrations of IFN-α checked byAb responses in the host (self /not self ??)

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A note of caution

Several features need to be investigated

4 issues:

which effector cell populations ?

how are they activated ?

how can they sense differentconcentrations of IFN-α ?

how do they propagate the signal ?

As many issues for future research !!

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Acknowledgements

Dr. Paolo Candotti, IZSLER, Brescia

Dr. Ivonne Laura Archetti, IZSLER, Brescia

Dr. Barbara Begni, IZSLER, Brescia

Lab technicians, IZSLER

Antonio Cristiano

Cinzia Mantovani

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Thank you forthe attention !