Does chronical deoxynivalenol-feeding modulate the immune

University of Veterinary Medicine Hannover

Does chronical deoxynivalenol-feeding modulate the immune

response in endotoxaemic pigs?

Inaugural-Dissertation

to obtain the academic degree

Doctor medicinae veterinariae

(Dr. med. vet.)

submitted by

Tanja Tesch

Gifhorn

Hannover 2017

Academic supervision: 1. Prof. Dr. med. vet. Gerhard Breves

Physiologisches Institut

Stiftung Tierärztliche Hochschule Hannover

2. Prof. Dr. Dr. Sven Dänicke

Institut für Tierernährung

Bundesinstituts für Tiergesundheit (Friedrich-

Loeffler- Institut) Braunschweig

1. Referee: Prof. Dr. med. vet. Gerhard Breves

2. Referee: Prof. Dr. Pablo Steinberg

Day of oral examination: 02.11.2017

This study was funded by the German Research Funding Organisation

(DFG-Projekt DA 558/1-4)

I would like to thank my family (Danke auch von Herzen an dich Omi) and all the

people in my life who encouraged and supported me in everything I do.

Above all, I would like to thank my mom from the bottom of my heart for her end-

less love and her strong faith in me.

If you can dream it, you can do it.

(Walt Disney)

Parts of this thesis have already been published, accepted or submitted for pub-

lication in the following journals:

T. Tesch, E. Bannert, J. Kluess, J. Frahm, S. Kersten, G. Breves, L. Renner, S. Kah-

lert, H.J. Rothkötter, and S. Dänicke

Does Dietary Deoxynivalenol Modulate the Acute Phase Reaction in Endotox-

aemic Pigs? - Lessons from Clinical Signs, White Blood Cell Counts, and TNF-

Alpha.

Toxins, 2016, Volume 8/3, DOI:10.3390/toxins8010003

T. Tesch, E. Bannert, J. Kluess, J. Frahm, L. Hüther, S. Kersten, G. Breves, L.

Renner, S. Kahlert, H.J. Rothkötter, and S. Dänicke

Relationships between body temperatures and inflammation indicators under

physiological and pathophysiological conditions in pigs exposed to systemic lip-

opolysaccharide (LPS) and dietary deoxynivalenol (DON)

Journal of Animal Physiology and Animal Nutrition, 2017, Volume 1/11, DOI:10.

1111/jpn.12684

Furthermore, results of this thesis were presented in form of oral presentations

or posters at the following conferences:

1. The oral deoxynivalenol exposition in pigs is modulating the patho-

physiological effect of a downstream LPS stimulus: methodological as-

pects

T. Tesch, E. Bannert, J. Kluess, J. Frahm, S. Kersten, S. Dänicke

Junior Scientist Symposium 2014, 19.-22.08.2014, Mariensee, Germany,

Proceedings p. 23

2. Effects of a lipopolysaccharide (LPS) stimulus in pigs chronically exposed

to dietary deoxynivalenol (DON): Clinical signs and leukocytes

37th Mycotoxin Workshop, 01.-03.06.2015, Bratislava, Slovakia, Proceed-

ings p. 145

3. Effects of a lipopolysaccharide (LPS) stimulus in pigs chronically exposed

to dietary deoxynivalenol (DON): Clinical signs and leukocytes

T. Tesch, E. Bannert, J. Kluess, J. Frahm, S. Kersten, G. Breves, L. Renner,

S. Kahlert, H.J. Rothkötter, and S. Dänicke

Junior Scientist Symposium 2015, 21.-23.09.2015, Züssow, Germany,

Abstract collection p.53

4. Auswirkungen eines Immunstimulus auf die Akute-Phase-Reaktion bei

Deoxynivalenol exponierten Schweinen



13. Schweine- und Geflügelernährungstagung, 24.-26.11.2015, Lu-

therstadt Wittenberg, Germany, Proceedings pp. 79-81.

5. Effects on the acute phase reaction (APR) in lipopolysaccharide (LPS)-

stimulated pigs pre-exposed with deoxynivalenol (DON)



70. Jahrestagung der Gesellschaft für Ernährungsphysiologie (GfE), 08.-

10.03.2016, Hannover, Germany, Proc. Soc. Nutr. Physiol. 25, p. 152

6. Relationships between body core temperature and indicators of systemic

inflammation induced by lipopolysaccharide (LPS) in dependence of an

oral exposure to deoxynivalenol (DON) in pigs

T. Tesch, E. Bannert, J. Kluess, J. Frahm, L. Hüther, S. Kersten, G. Bre-

ves, L. Renner, S. Kahlert, H.J. Rothkötter, and S. Dänicke

38th Mycotoxin Workshop, 02.-04.05.2016, Berlin, Germany, Proceedings

p. 138

7. Is the relationship between abdominal and rectal temperature constant in

healthy and lipopolysaccharide-stimulated, deoxynivalenol-fed pigs?

T. Tesch, E. Bannert, J. Kluess, J. Frahm, S. Kersten, G. Breves, L. Ren-

ner, S. Kahlert, H.J. Rothkötter, and S. Dänicke

24th International Pig Veterinary Society Congress (IPVS) and 8th Euro-

pean Symposium of Porcine Health Management (ESPHM), 07.-

10.06.2016, Dublin, Irland, Proceedings p. 635

8. Relationship between body temperature and systemic inflammation

markers induced by lipopolysaccharide in dependence of an oral deoxyni-

valenol exposure

T. Tesch, E. Bannert, J. Kluess, J. Frahm, L. Hüther, S. Kersten, G. Bre-

ves, L. Renner, S. Kahlert, H.J. Rothkötter, and S. Dänicke

24th International Pig Veterinary Society Congress (IPVS) and 8th Euro-

pean Symposium of Porcine Health Management (ESPHM), 07.-

10.06.2016, Dublin, Irland, Proceedings p. 618

Contents

TABLE OF CONTENT

Introduction - 1 -

Background - 3 -

1. Deoxynivalenol

1.1. Source and occurrence

1.2. Structure and physical-chemical properties

1.3. Metabolism and toxicokinetics of DON

1.3.1. Intake and excretion

1.4. Mode of action and toxicity

1.4.1. Clinical signs of acute and subacute intoxication

1.4.2. Clinical signs of chronic and subchronic intoxication

1.4.3. Effects on blood parameters

1.4.4. Modulation of the immune system

2. Lipopolysaccharide



2.3. Mode of action

2.4. Early acute phase response and its effects on immune parameters

3. Interactions between DON and LPS

Scope of the thesis - 22 -

Paper I - 24 -

Does dietary deoxynivalenol modulates the acute phase reaction in endo-

toxaemic pigs? - Lessons from clinical signs, white blood cell counts and

TNF-alpha –

Paper II - 50 -

Relationships between body temperatures and inflammation indicators under

physiological and pathophysiological conditions in pigs exposed to systemic

lipopolysaccharide (LPS) and dietary deoxynivalenol (DON)

General discussion - 76 -

Summary - 91 -

Contents

Zusammenfassung - 94 -

References - 97 -

Abbreviations

Abbreviations

ALB albumin

ALAT alanine-aminotransferase

AP alkaline phosphatase

AP-1 activating protein 1

APP acute phase protein

APR acute phase reaction

ASAT aspartate-aminotransferase

BE base excess

BGA blood gas analysis

BW body weight

CD-14 cluster of differentiation 14

C/EBP CCAAT enhancer-binding protein

COX-2 cyclooxygenase 2

CREA creatinine

CRP c-reactive protein

DON deoxynivalenol

ED50 effective dose 50 (amount of a substance required to produce a specific

effect in half of an animal population)

GIT gastrointestinal tract

GLDH glutamate dehydogenase

GLU glucose

Ɣ-GT gamma glutamyl transferase

HCT haematocrit

HGB haemoglobin

HPA axis hypothalamic-pituitary-adrenal axis

IDO indoleamine 2,3-dioxygenase

IFNy interferon gamma

IgA immunoglobulin A

IL-1, -1ß, 2, 6, 8 interleukin 1, 1ß, 2, 6, 8

iNOS inducible nitric oxide synthase

IRF3 interferon regulatory factor 3

Abbreviations

i.v. intra venous

KDO 2-keto-3-deoxyoctonic acid

KYN kynurenine

KYN-TRP ratio kynurenine to tryptophan ratio

LAC lactate

LBP lipopolysaccharide binding protein

LD lethal dose (amount of a substance that is likely to cause death

to an animal)

LDH lactate-dehydrogenase

LPS lipopolysaccharide

MAPk mitogen-activated protein kinases

MCH mean corpuscular haemoglobin

MD-2 myeloid differentiation protein 2

mRNA messenger ribonucleic acid

MyD88 Myeloid differentiation factor 88

NFκB Nuclear factor kappa-light-chain-enhancer of activated B

cells

NO nitric oxide

PAF platelet activating factor

PAMP pathogen-associated molecular pattern

pCO2 carbon dioxide partial pressure

PGE2 prostaglandin E2

pH negative logarithm of the hydrogen ion concentration

p.i. post infusionem

pig-MAP pig-mitogen activated protein

pO2 oxygen partial pressure

PRR pathogen recognition receptor

RBC red blood cell

RNI reactive nitrogen intermediate

ROI reactive oxygen intermediate

SAA serum amyloid A

TB total bilirubin

TDO tryptophan 2,3-dioxygenase

Abbreviations

TLR4 toll-like receptor 4

TNF-α tumor necrosis factor alpha

TP total protein

TRP tryptophan

TXA thromboxane

WBC white blood cell

Introduction

- 1 -

Introduction

Under healthy conditions the organism is situated in homeostasis, keeping its

vital functions, such as body core temperature and different blood parameters

stable within their physiological limits. This inner balance is maintained against a

large number of intrinsic and extrinsic influences by self-regulatory mechanisms,

ensuring an adequate supply and function of all organs and tissues. In patho-

physiological conditions, the organism initiates an immune response in order to

eliminate the source of irritation rapidly, self-regulatory mechanisms get impaired

causing a dysregulation of homeostasis in several body functions. In both,

healthy and pathophysiological conditions, the liver, as central organ of metabo-

lism, detoxification and immune response, is of crucial interest for keeping or

rather restoring homeostasis.

In swine production one of the factors disturbing homeostasis is a chronic dietary

mycotoxin exposure that a large proportion of animals frequently encounter dur-

ing their lifetime. One of the most frequently detected mycotoxin in toxicologically

relevant levels is the B-trichothecene DON. This toxin is mainly produced by the

fungi Fusarium graminearum and F. culmorum and can be found in cereal

grains, especially wheat and maize, which are major portions of pig’s diets.

Swine are known as the most DON-sensitive species (Pestka and Smolinski

2005, Döll and Dänicke 2011) and symptoms such as vomiting, inappetence,

and reduced weight gain are often related to DON-contamination of their feed.

In addition to DON, the infectious pressure in the swine production site also con-

tains further influencing factors like a co-exposure to LPS-releasing Enterobacte-

riaceae such as Salmonella, Campylobacter or E.coli (Smith and Halls 1968,

Berends, Urlings et al. 1996). The endotoxin LPS is a component of the outer cell

membrane of gram-negative bacteria and has a high stimulatory potential for the

innate and acquired immune response characterized by its interaction with dif-

ferent types of leukocytes and their subsequent biosynthesis of various effector

molecules, such as TNF-α, one of the major fever inducing and amino acid me-

tabolism altering cytokines in mammals (Raetz and Whitfield 2002). Moreover

these acute internal restoration processes of the organism to LPS are accompa-

nied by clinically observable indicators of an immune response, such as the car-

Introduction

- 2 -

dinal symptom fever (Dänicke, Brosig et al. 2013), while a chronic stimulation of

the immune system with DON, can comprise slowly initiated and less obvious

modifications in different measurable immune parameters, such as increased

leukocyte counts (Rotter, Thompson et al. 1994, Chaytor, See et al. 2011), pro-

inflammatory cytokines, chemokines and other immune related proteins (Dong,

Azcona-Olivera et al. 1994, Azcona-Olivera, Ouyang et al. 1995, Azcona-

Olivera, Ouyang et al. 1995, Ouyang, Azcona-Olivera et al. 1995).

Additionally synergistic effects between DON and LPS has been reported in the

induction of pro-inflammatory cytokine expression (Islam and Pestka 2003, Islam

and Pestka 2006) as well as in potentiation of their toxicity (Döll, Schrickx et al.

2009, Döll, Schrickx et al. 2009) during the APR. Thus, the immune response to

LPS in animals pre-exposed to DON is reflected in altered blood parameters and

clinical manifestation (Raetz and Whitfield 2002).

In veterinary practice, the clinical examination and rectal temperature are giving

the first and easiest observable information for pathophysiological changes in

animal´s health conditions. But especially rectal temperature measurement holds

a high source of error and although measured rectal temperature is regarded to

represent actual body core temperature, it has to be emphasized that the rectum

per se does not belong to the body core strictly (Greenes and Fleisher 2004,

Hanneman, Jesurum-Urbaitis et al. 2004). Therefore, in case of pathophysiologi-

cal conditions, rectally measured temperature may not reflect changes in body

core homeostasis respectively porcine´s health status compared to in-

traabdominal core temperature and inflammatory blood parameters.

Background

- 3 -

Background

1. Deoxynivalenol


DON is a naturally occurring mycotoxin, a secondary metabolite that is mainly

produced by filamentous fungi, which are growing in agricultural products on the

field or during storage. It is formed by several genera of fungi such as Cephalo-

sporum, Fusarium, Myrothecium, Stachybotrys, Trichoderma, Trichothectum,

Verticimonosporium in which Fusarium species are the most important produc-

ers of DON (EFSA 2004, Rocha, Ansari et al. 2005). Fusarium species primarily

infect wheat, barley, oats, rye and maize and thus lead to economic losses in

agriculture production (McLean 1996, EFSA 2004, Abbas, Yoshizawa et al.

2013). Whereby the fungal growth and the production of toxin is affected by vari-

ous factors, including temperature, air humidity, rainfall during anthesis and corn

harvest, soil treatment, crop rotation and plant stressors such as draught or

over-irrigation, insect damage and pesticide exposure (Fink-Gremmels 1999,

Oldenburg, Valenta et al. 2000). In northern temperate regions, especially F.

graminearum and F. culmorum are the mainly occurring fungi of the Fusarium

family, with DON as their most frequently detected toxin in grain and feedstuff

(Chelkowski 1998, EFSA 2004). In addition to its high phytotoxicity, the mycotox-

in contamination of cereal related products including animal feed also causes

several adverse effects, e.g. health risks, in livestock farming accompanied by

economic losses (Rotter, Prelusky et al. 1996, EFSA 2004, Rocha, Ansari et al.

2005).


DON is one of the trichothecene mycotoxins, a large family comprising over 200

chemically closely related toxins. Trichothecenes are amphipathic (hydrophilic

and lipophilic), low-molecular-weight (200-500 Da) tetracyclic sesquiterpenoids,

characterized by a common core structure (Cole 2003, Grove 2007) with an

epoxide ring at C12 and C13 as well as a double bond at position C9 and C10.

This epoxide configuration is responsible for their toxicity and ensures that the

Background

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molecule is chemically stable. Based on different substituents, side-chains and

thereby chemical properties, trichothecenes are classified in four subclasses

(Tab. 1) (Ueno, Sato et al. 1973, Ueno 1985).

Table 1: Trichothecene sublasses

Figure 1: Chemical structure of 12,13-Epoxy-3,4,15-trihydroxy- trichothec-9-en-8-on (C15H20O6),

also known as DON.

DON belongs to the Type B trichothecenes and is chemically designed as 12,13-

Epoxy-3,4,15-trihydroxy-trichothec-9-en-8-on (Fig. 1), a colorless, crystalline sol-

id with a molar mass of 296.32 g/mol. It endures temperatures ranging from 170

to 350°C and is stable in neutral as well as in acid pH values, thus this mycotox-

in resists animal feed or food processing and boiling (Larsen, Hunt et al. 2004,

Sobrova, Adam et al. 2010).

1.3. Metabolism and toxicokinetics of DON

DON is present in many cereals respectively cereal products, as already men-

tioned, and has shown different toxic effects on mammals. For the protection of

Classification Characteristics Toxins

Type A

• hydroxyl group, ester function or no

oxygen substitution at C-8;

• oxygen function (hydroxyl or acetyl

group) at C-3 in Fusarium toxins;

• more toxic than Type B

Calonectrin, 4,15-Diacetoxyscirpenol, 7,8-

Dihydroxy-Calonectrin, Harzianum A, HT-2

Toxin, Isotrichodermol, Mono-

acetoxyscirpenol, Neosolaniol, T-2 Toxin,

Trichodermin, Trichodermol

Type B

• carbonyl (keto) function at C-8;

• hydroxyl group at C-7 and oxygen

function (hydroxyl or acetyl group) at C-

3 in Fusarium toxins;

• less toxic than Type A

3-Acetyldeoxynivalenol, 15-Acetyl-

deoxynivalenol, Deoxynivalenol,

Fusarenon X, Nivalenol, Trichothecin,

Trichothecinol A

Type C • epoxide group at C-7, C-8; Baccharin, Crotocin

Type D • additional ring linking C-4 and C-15;Myrotoxin B, Roridin A, Satratoxin H,

Verrucarin A

Background

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animals and humans the Commission of European Communities established a

so called guidance values for critical DON concentrations in feed material and

complete feeds (2006/576/EC) based on the recommendation of the European

Food Safety Authority (EFSA 2004).

The guidance value in mg/kg relative to a feeding stuff with a moisture content of

12% is accordingly defined as follows: for instance maize by-products 12 mg

DON/kg, complementary and complete feeding stuffs for pigs 0.9 mg DON/kg and

for calves, lambs and kids 2 mg DON/kg (annex guidance values 2006/576/EC).

1.3.1. Intake and excretion

Animals are usually exposed to DON by the consumption of contaminated

feedstuff. Due to the molecular size and amphipathic properties, DON can move

passively across cell membranes and can be easily absorbed via the integumen-

tary and gastrointestinal system (Sulyok, Krska et al. 2007). But DON´s metabo-

lism and toxicokinetics differs within animal species, thus the distinct difference

in toxin sensitivity, whereby pigs are known as the most susceptible species,

followed by rodents > dogs > cats > poultry > ruminants (Pestka and Smolinski

2005), could be explained. However, metabolites of DON that are undetectable

by conventional analytical techniques (modified mycotoxins) can also cause an

apparently altered sensitivity due to the incorrectly determined mycotoxin con-

centration (Rychlik, Humpf et al. 2014).

In pigs the bioavailability of ingested DON was shown to vary between 82 - 110%

(Rohweder, Kersten et al. 2013), in contrast to sheep that showed a much lower

systemic absorption of 6 - 10% (Prelusky, Veira et al. 1986, Prelusky, Veira et al.

1987), as well as dairy cows and laying hens with data < 1% (Prelusky,

Trenholm et al. 1984, Prelusky, Hamilton et al. 1986).

After oral consumption DON is rapidly absorbed in the proximal parts of the

swine´s small intestine, thus it can be detected after 15 - 20 min in plasma with

the maximal concentration between 0.8 - 4.1 h (Dänicke, Valenta et al. 2004,

Goyarts and Dänicke 2006, Rohweder, Kersten et al. 2013).

The detoxification of DON takes place on the one hand by de-epoxidation,

wherein the C12,13 epoxide group is split off by microorganism in the digestive

Background

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tract, reaching nearly 100% de-epoxidation in the rectal faeces (Rotter, Prelusky

et al. 1996, Dänicke, Valenta et al. 2014) and on the other hand by conjugation

with glucuronic acid and sulfation by the liver (Ritter 2000, Gamage, Barnett et

al. 2006, Dänicke and Brezina 2013). Besides the faecal excretion, the main part

of DON and its metabolites are excreted by the kidneys in the urine.

Figure 2: Metabolism (conversion of the native toxin to various degradation derivates) of DON in

the pig: DON contaminated feed passes the stomach (1.) and enters the small intestine (2.),

where the toxin is absorbed and drained via the portal vein to the liver (3.). From there, one part

of DON goes via the bile back into the small intestine (enterohepatic circulation) and is fractionally

excreted by faeces, while the other part reaches the blood circulation and is excetred by the

urine (4.).

Toxic residues in pigs, chronically exposed to 0 - 1.23 mg DON/kg BW for 11

weeks, were found with highest concentrations in bile (144 ng/mL), followed by

kidneys > serum > liver (3 ng/mL) and muscles (Döll, Dänicke et al. 2008).

1.4. Mode of action and toxicity

Various effects of DON on the mammal´s organism were described. These ef-

Background

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fects include, besides feed refusal, reduced weight gain and vomiting, influences

on the protein metabolism, such as protein synthesis inhibition, and modulatory

effects on the immune system, including immune activation as well as suppres-

sion, depending on dosage and administration (Pestka and Smolinski 2005).

1.4.1. Clinical signs of acute and subacute intoxication

Characteristic signs for an acute intoxication with DON are feed refusal, weight

loss, salivation, vomitus and diarrhea (Young, McGirr et al. 1983, Prelusky and

Trenholm 1993, EFSA 2004, Pestka and Smolinski 2005) . In young swine ano-

rexia was observed from a concentrations of 1 - 2 mg DON/kg feed (Prelusky

1996), while the minimal oral emetic dose in pigs is indicated between 0.05 - 0.2

mg DON/kg BW (Larsen, Hunt et al. 2004). However, the ED50 values ranges be-

tween 0.085 - 0.088 mg purified DON/kg BW and 0.02 mg i.v. injected DON/kg

BW (Young, McGirr et al. 1983, Prelusky and Trenholm 1993). The exposure of

very high doses of DON could also cause shock-like death, observed in mice

with the LD 50 of 78 mg orally DON/kg BW or rather 49 mg i.p. injected DON/kg

BW (Pestka and Smolinski 2005).

1.4.2. Clinical signs of chronic and subchronic intoxication

Pigs, exposed to a sustained feeding of a DON contaminated diet, generally

showed decreased live weight gain (Bergsjo, Matre et al. 1992, Bergsjo,

Langseth et al. 1993, Dänicke, Valenta et al. 2004, Pestka and Smolinski 2005)

due to anorexia with a reduction in voluntary feed intake by 5.4% per 1 mg

DON/kg feed (Dänicke, Döll et al. 2008) up to complete refusal at concentrations

of 12 mg DON/kg diet (Young, McGirr et al. 1983).

1.4.3. Effects on blood parameters

In clinical-chemical parameters increasing values of ALB concentrations were

observed in mice, feed with 1 mg DON/kg BW for five weeks as well as α1- and

α2-globulin decreased concurrently (Tryphonas, Iverson et al. 1986). These re-

sults were confirmed in pigs exposed to 1 - 3 mg orally DON/kg diet (Prelusky,

Gerdes et al. 1994), additionally with arising increased levels of albumin/globulin

ratio (Rotter, Thompson et al. 1994). But also contrasting data have been re-

Background

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ported, showing an increase in globulin levels (Chaytor, See et al. 2011) in pigs

feed with a diet, containing 0.9 mg DON/kg feed, or decreased values in TP

(Trenholm, Foster et al. 1994, Döll, Dänicke et al. 2003) and ALB (Bergsjo,

Langseth et al. 1993, Grenier, Loureiro-Bracarense et al. 2011) after exposure to

DON contaminated diets with concentrations between 3 and 3.9 mg DON/kg

feed. Additionally some studies in pigs, fed with a naturally contaminated diet up

to 6.8 mg DON/kg feed, were not able to observe any alterations in plasma TP or

ALB levels (Cote, Beasley et al. 1985, Goyarts, Dänicke et al. 2005, Kullik,

Brosig et al. 2013) although fractional and total synthesis rate of ALB decreased

significantly (Goyarts and Dänicke 2005, Kullik, Brosig et al. 2013).

Besides these proteins, other liver specific enzymes or values are also of inter-

est in cases of intoxication with Fusarium toxins. Regarding to this, increased

levels of AP and Cholesterol were found in pigs exposed to a DON contaminated

diet with 0.9 mg DON/kg feed (Chaytor, See et al. 2011). While other studies re-

ported a decrease in AP (Trenholm, Foster et al. 1994) after consumption of 3.4

mg DON/kg feed but also in GLDH (Döll, Dänicke et al. 2003) after feeding up to

3.9 mg DON/kg diet. Nevertheless there are also data in pigs, fed with a DON

contaminated diet (5.8 - 6.8 mg DON/kg diet), showing no significant alterations

in AP, ASAT, ALAT, TB, Cholesterol, CREA, ɣ-GT, GLDH, LDH, (Cote, Beasley et

al. 1985, Goyarts, Dänicke et al. 2005).

Further blood parameters are summarised in the red blood count, that is show-

ing increasing values in RBC, HCT, HGB and Platelets as well as concurrently

decreasing levels in MCH (Prelusky, Gerdes et al. 1994) in swine exposed to 1 -

3 mg DON/kg diet. Contradictory results with a decrease in HCT, HGB and RBC

(Arnold, Karpinski et al. 1986, Arnold, McGuire et al. 1986) were observed in rats

and mice fed with DON contaminated feed rations (6.25 mg DON/kg feed). Addi-

tionally decreased HGB levels were also found in pigs after consumption of 3.5

mg DON/kg diet (Bergsjo, Langseth et al. 1993). Even though the majority of

studies in DON fed pigs (2.5 - 4.6 mg DON/kg diet) cannot confirm any significant

alterations in the red blood count (Pinton, Accensi et al. 2008, Grenier, Loureiro-

Bracarense et al. 2011, Bannert, Tesch et al. 2015).

Nonetheless, in total it has to be underlined that most of the mentioned studies

Background

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were not able to distinguish between the directly toxic effects of DON and the

indirect effects based on the reduced voluntary feed intake and related nutrient

deficiency.

1.4.4. Modulation of the immune system

The immune-modulatory properties of DON, which can either be immune stimula-

tory or immune suppressive (Rotter, Prelusky et al. 1996, Pestka, Zhou et al.

2004, Pestka 2008), are linked to the mentioned inhibitory effects on protein syn-

thesis and depending on dosage regime as well as the type of administration.

White blood cells are the functional cells of the immune system and thus a pri-

mary target for exotoxins such as DON (Arnold, McGuire et al. 1986, Forsell,

Jensen et al. 1987, Pestka, Zhou et al. 2004). The immune suppressive capaci-

ties of DON generate apoptosis and cytotoxicity in leukocytes, macrophages, T-

and B-lymphocytes with relation to MAPk phosphorylation in rodent and in vitro

studies (Pestka, Yan et al. 1994, Islam, Nagase et al. 1998, Yang, Jarvis et al.

2000, Islam, King et al. 2003). Further published data showed necrosis and at-

rophy of cells or tissues with high turnover rates, such as bone marrow, intestinal

mucosa, lymph nodes and spleen (Bondy and Pestka 2000, Pestka, Islam et al.

2008). These immune suppressive implications of DON were also confirmed in

pigs, showing leukopenia after infusion of 0.1 mg DON/kg BW (Kluess, Kahlert et

al. 2015) and apoptosis in lymphoid tissues as well as in hepatocytes after i.v.

injection of 1 mg DON/kg BW (Mikami, Yamaguchi et al. 2010). Contrasting re-

sults showed no alterations in expression and phosphorylation of p38 MAPk in

swine fed with 3 – 10 mg DON/kg feed while in vitro data of the same study re-

vealed a decreased expression of p38 MAPk without influence on the overall

phosphorylation of this kinase (Wollenhaupt, Dänicke et al. 2006).

The reduced number of circulating white blood cells consequently increased the

predisposition to disease-causing pathogens (Bondy and Pestka 2000) and inhi-

bition of antibody responses or impaired delayed-type hypersensitivity (Rotter,

Thompson et al. 1994, Rotter, Prelusky et al. 1996, Overnes, Matre et al. 1997).

On the other hand the immune stimulatory effects of DON have been proven by

an increase in leukocyte counts in rats and mice exposed to 6.25 mg DON/kg

feed (Arnold, Karpinski et al. 1986, Arnold, McGuire et al. 1986) as well as in

Background

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pigs after feeding a diet containing 0.8 - 3 mg DON/kg (Rotter, Thompson et al.

1994, Chaytor, See et al. 2011) or rather after an acute i.v. injection of 1 mg

DON/kg BW (Mikami, Kubo et al. 2011).

In addition to these direct effects on blood cells, DON was shown to increase the

production and secretion of pro-inflammatory cytokines, chemokines and other

immune related proteins (Dong, Azcona-Olivera et al. 1994, Azcona-Olivera,

Ouyang et al. 1995, Azcona-Olivera, Ouyang et al. 1995, Ouyang, Azcona-

Olivera et al. 1995). For instance, an elevated IgA production by B-Lymphocytes

in mice was detected after feeding 25 mg DON/kg diet (Dong and Pestka 1993,

Pestka, Zhou et al. 2004). Thereby also T-Lymphocytes and macrophages were

involved, due to the fact that DON is incapable to directly induce IgA secretion in

primary B-cells (Warner, Brooks et al. 1994). However the DON caused up-

regulation of pro-inflammatory cytokines, which were released by T-lymphocytes

and macrophages, is probably crucial for B-cell differentiation to plasma cells

and therewith for IgA secretion (Pestka, Zhou et al. 2004). Also an enhanced

stability of cytokine mRNA for COX-2, IL-6 and TNF-α (Wong, Schwartz et al.

2001, Chung, Zhou et al. 2003, Moon and Pestka 2003) as well as an increase

in immune associated genes on the transcriptional and translational level is re-

peatedly reported in rodents and different cell lines (Moon and Pestka 2003,

Moon and Pestka 2003, Kinser, Jia et al. 2004, Nogueira da Costa, Mijal et al.

2011, van Kol, Hendriksen et al. 2011, He, Pan et al. 2013). Regarding to the

translational level the so called superinduction by protein synthesis inhibitors like

DON, is probably based on the inhibition of labile repressor protein, that in turn

cause an activation of kinases such as p38 and MAPk (Yang, Jarvis et al. 2000,

Zhou, Islam et al. 2003, Pestka, Zhou et al. 2004). On the transcriptional level

DON induced an increase of binding activities in transcriptional factors, such as

AP-1, NFκB and C/EBP, in the spleen of mice (Zhou, Islam et al. 2003).

These immune stimulatory effects of DON were also verified in swine, showing

increased levels of IL-1, IL-6, IL-8 and TNF-α after being exposed to 0.8 mg

DON/kg diet or rather an acute i.v. injection of 1 mg DON/kg BW (Chaytor, See et

al. 2011, Mikami, Kubo et al. 2011). Furthermore, an increase of mRNA expres-

sion of IL-1ß, IL-2 and IL-6 (jejunum) respectively IL-1ß, IL-6 and TNF-α (ileum) in

Background

- 11 -

the small intestine were found in pigs, consuming a naturally contaminated diet

with 3 mg DON/kg feed (Bracarense, Lucioli et al. 2012). Besides these results

an increase of pro-inflammatory cytokines has also been detected in different

porcine cell line studies, showing improved values of mRNA expression in alveo-

lar macrophages and hepatocytes (Döll, Schrickx et al. 2009, Döll, Schrickx et al.

2009). Contrarily, a study in pigs infusing 100 µg DON/kg BW for 60 min could

not show any increase in pro-inflammatory cytokines in serum (Dänicke, Brosig

et al. 2013).

All immune activating effects considered that DON improves the resistance of the

organism against pathogens such as LPS (Pestka, Zhou et al. 2004).

2. Lipopolysaccharide


Gram-negative bacteria, such as Enterobactericteriaceae, are ubiquitously pre-

sent in the environment as well as being a part of the commensal gastrointesti-

nal microbiota in animals and humans (Berczi, Bertok et al. 1966, Raetz and

Whitfield 2002). The outer layer of these bacterial cell membranes is largely

covert with an asymmetric phospholipid bilayer membrane, the so called LPS,

that is essential for the membrane properties as permeability barrier and bacteri-

al vitality (Sperandeo, Deho et al. 2009). Moreover LPS is acting as endotoxin

when released from the surface in case of bacterial multiplication and death

(Hewett and Roth 1993, Rietschel, Kirikae et al. 1994). Free LPS induces a

strong immune response in mammals and triggers the systemic reaction of the

immune system in case of infections caused by gram-negative bacteria or cer-

tain pathophysiological conditions that enables an intensified intestinal absorp-

tion of LPS from gut-derived bacteria (Hewett and Roth 1993, Schletter, Heine et

al. 1995, Aderem and Ulevitch 2000, Caroff, Karibian et al. 2002).


LPS consists of three covalently linked main parts: a hydrophobic domain as the

Background

- 12 -

source of toxicity (lipid A), a core region (oligosaccharides) and a hydrophilic O-

specific chain responsible for most antigenic properties (polysaccharide).

The lipid A domain is the most invariable part of LPS among all components. It is

an amphipathic layer with a hydrophobic anchor in the phospholipid membrane

consisting of saturated fatty acids in varying number, location and chain length

and on the other side a hydrophilic connection to the LPS core region formed by

disaccharides of glucosamine (Galanos, Luderitz et al. 1985, Rietschel, Kirikae

et al. 1994, Zähringer, Lindner et al. 1994, Raetz and Whitfield 2002). Variations

in the lipid A structure among different bacterial genera appear in both, fatty ac-

ids and disaccharides that can be substituted by acyl or rather phosphoryl

groups. Finally the lipid A domain is necessary for bacterial viability (Schletter,

Heine et al. 1995, Alexander and Rietschel 2001) and mainly responsible for the

toxic activity and immune modulating properties of LPS (Zähringer, Lindner et al.

1994, Raetz and Whitfield 2002).

Thereon follows the core oligosaccharide region of LPS comprising 10 to 12 units

of saccharides and that is subdivided into an inner and outer core (Caroff,

Karibian et al. 2002, Erridge, Bennett-Guerrero et al. 2002). The inner portion is

mainly formed by units of the acidic sugar 2-keto-3-deoxyoctonic acid and L-

glycero-D-manno configured heptose and generates the linkage to the lipid A

region (Raetz and Whitfield 2002), whereby KDO and lipid A form the minimal

LPS structure. In contrast, the outer core contains mainly hexoses such as D-

glucose, D-galactose and N-acetyl-D-glucosamine in an intermediate structural

diversity and constitutes the linkage to the o-specific chain as the third main part

of LPS (Schletter, Heine et al. 1995, Raetz and Whitfield 2002). Both inner and

outer core portions are usually substituted by phosphate, 2-aminoethyl-

phosphate or hexose residues that stabilise core´s structure by binding divalent

cations (Rietschel, Kirikae et al. 1994, Raetz and Whitfield 2002, Holst 2011).

The subsequent and most external part of the gram-negative bacteria cell wall is

the O-specific or polysaccharide chain that defines the bacterial serotype and

serves as important surface antigen (Knirel and Kochetkov 1994, Schletter,

Heine et al. 1995). This domain is made up of a polymer of repeating oligosac-

Background

- 13 -

charide units the most variable fragment of LPS (Rietschel, Kirikae et al. 1994,

Erridge, Bennett-Guerrero et al. 2002, Raetz and Whitfield 2002).

Figure 3: The common chemical structure of lipopolysaccharide of gram-negative bacteria.

2.3. Mode of action

Mammal´s organism responses very sensitive to LPS intoxication, whereby vari-

ous signalling pathways can be initiated to activate the immune system. In this

context the activation of the transmembrane TLR4 with its extracellular, ligand-

binding and intracellular, signal transducing domain, is of particular importance

(Rock, Hardiman et al. 1998, Rallabhandi, Bell et al. 2006, Jin and Lee 2008).

LPS molecules that reach the blood circulation are captured by LPB and subse-

quently transported to CD14 on the surface of monocytes, macrophages or poly-

morphonuclear leukocytes (Thomas, Kapoor et al. 2002). This trimolecular LPS-

binding complex initiates an alteration in the confirmation of TLR4 caused by MD-

2, thus a direct contact between the lipid A domain of LPS and TLR4 is generated

(Medzhitov and Janeway 1997, Medzhitov and Janeway 1997, Poltorak,

Ricciardi-Castagnoli et al. 2000, Correia, Soldau et al. 2001, Visintin, Latz et al.

2003, Park, Song et al. 2009). After this activation of TLR4 two major intracellular

signal cascades exist: the early MyD88-dependent and the delayed MyD88-

indpendent response (Hoebe, Du et al. 2003, Yamamoto, Sato et al. 2003,

Yamamoto, Sato et al. 2003, Palsson-McDermott and O'Neill 2004, Bode,

Ehlting et al. 2012).

The MyD-88-dependent pathway activates the transcription factors NFκB ulti-

mately by involving various sequences of adaptor proteins and signalling inter

mediates (Geng, Zhang et al. 1993, Takeda and Akira 2004), while the MyD88-

Background

- 14 -

Figure 4: Summarised schematic illustration of the LPS signalling and APR induction with related

immunological and metabolic effects in mammals.

indpendent pathway induces IRF3 as well as a delayed NFκB response (Akira

2001, Doyle, Vaidya et al. 2002, Zhai, Shen et al. 2004). The activated transcrip-

tion factor NFκB in turn translocates to the nucleus and triggers the expression of

genes, encoding pro-inflammatory cytokines such as IL-1β, IL-6 and IL-8, IFN-γ,

Background

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COX-2 and iNOS (Medzhitov and Janeway 1997, Medzhitov and Janeway 1997,

Palsson-McDermott and O'Neill 2004, Takeda and Akira 2004, Bode, Ehlting et

al. 2012). Thus the new gene transcription ensures the release of a massive

amount of pro-inflammatory cytokines and enzyme products as well as a coordi-

nated early APR (Elsbach 2000, Raetz and Whitfield 2002, Palsson-McDermott

and O'Neill 2004, Gruys, Toussaint et al. 2005, Bode, Ehlting et al. 2012).

2.4. Early acute phase response and its effects on immune parameters and

tissues

The APR is an immediate, non-specific, non-adaptive defence response of the

innate immune system to reconstitute homeostasis as soon as possible (Gruys,

Toussaint et al. 2005). Potential triggers of an APR induction like bacterial infec-

tion, trauma, neoplasms and other cell damage release a wide range of marked

metabolic, endocrine and immunological alterations such as fever, anorexia, de-

pression, leucocytosis and increased protein catabolism in a chronological order

(Ceciliani, Giordano et al. 2002, Humphrey and Klasing 2004, Gruys, Toussaint

et al. 2005). The manifestation of the mentioned alterations is depending on the

severity of LPS exposure, thus a low-dose of endotoxin is known to stimulate the

immune response and result in an elimination of invading PAMPs while a higher

LPS load evokes tissue injury via activation of leukocytes and intravascular co-

agulation up to massive cell death and organ failure (Roth, Su et al. 1998).

For experimental purpose such as model for human infection disease the APR in

swine can be induced by local or systemic LPS application (Schrauwen and

Houvenaghel 1984, Meurens, Summerfield et al. 2012, Mair, Sedlak et al. 2014,

Wyns, Plessers et al. 2015).

At the site of infection, that can be located in the blood circulation as well as in

tissue, target cells such as macrophages and monocytes are activated through

the interaction of PRR and PAMPs (van Miert 1995, Medzhitov and Janeway

2000). This causes the release of diverse mediators including pro-inflammatory

cyto- and chemokines (e.g. IL-1β, IL-6, IL-8, TNF-α), lipid-derived factors (e.g.

PAF, TXA. PGE2), vasoactive substances (e.g. histamine, prostaglandins, leuko-

trienes, NO), pathogens directly killing RNI and ROI as well as pain promoting

Background

- 16 -

mediators such as bradykinin and serotonin (Blackwell and Christman 1996,

Alexander and Rietschel 2001, Tizard 2012). Due to the impact of especially cy-

to- and chemokines further leukocytes are attracted from peripheral blood and

lymphoid organs to enter the site of infection and sustain the inflammatory re-

sponse (Baumann and Gauldie 1994, Baggiolini 1998, Luster 1998, Dauphinee

and Karsan 2006).

In case of local tissue infection, circulating leukocytes, mainly neutrophils and

monocytes, interact with released vasoactive substances such as Il-8, NO, PGE2

and TNF-α, that activate endothelial expression of adhesion, integrin and chemo-

tactic molecules, dilate blood vessels, decelerate blood flow and increase vascu-

lar permeability (Strieter, Kunkel et al. 1989, Lamas, Michel et al. 1991, Harris,

Padilla et al. 2002, Gruys, Toussaint et al. 2005, Dauphinee and Karsan 2006,

Ley, Laudanna et al. 2007). Furthermore complement factors stimulate phagocy-

tosis activity via serum-mediated opsonisation of bacteria, serve as attractant for

chemotaxis and are cable of lysing bacteria by first-line killing as well as they

enhance the T- and B-cell immunity (Alexander and Rietschel 2001, Carroll

2004, Rus, Cudrici et al. 2005). In consequence of these early inflammatory ac-

tivities, white blood cells, thrombocytes, and erythrocytes leave the circulation

thus most importantly a severe systemic leukopenia occurs during the first 6 h

after APR induction (Solling, Nygaard et al. 2011, Stadler, Le et al. 2011, Kluess,

Kahlert et al. 2015), followed by slowly normalisation within the following 12 to

24 h (Copeland, Warren et al. 2005, Taudorf, Krabbe et al. 2007, Kluess, Kahlert

et al. 2015). Leukopenia is mostly based on neutropenia and lymphopenia as

neutrophils, being part of the innate immune system, make up the major propor-

tion of leukocytes and perform phagocytosis, while lymphocytes built the second

major group of leukocytes and generate the cell mediated (T-cell) or humoral (B-

cell) response of the adaptive immune system. The cellular migration-related

decrease of vascular tone additionally contribute to local edema in the area of

infected tissue (Baumann and Gauldie 1994).

Pro-inflammatory cytokines are rapidly cleared from the blood circulation (Gruys,

Toussaint et al. 2005), but IL-6 directly and glucocorticoids via HPA axis indirect-

ly induced the production of specific plasma proteins for sustaining APR (Amrani,

Background

- 17 -

Mauzy-Melitz et al. 1986, Klasing and Johnstone 1991, Baumann and Gauldie

1994). These so-called APPs mediate opsonisation of bacteria, chemotaxis of

white blood cells and bind molecules such as plasma iron and zinc to prevent

their uptake by microbes. APPs are mainly synthesised by hepatocytes. There-

fore the liver or rather the KUPFFER cells are of particular importance in clearing

LPS from circulation (Hewett and Roth 1993). Additionally an extrahepatic APP

response is also observable in other cells and tissues such as lymph nodes, ton-

sils, spleen, white blood cells and intestinal epithelial cells (Uhlar and Whitehead

1999, Vreugdenhil, Dentener et al. 1999, Skovgaard, Mortensen et al. 2009).

APP are divided in two groups: so called positive APP and negative APP (Morley

and Kushner 1982, Klasing and Johnstone 1991, Gruys, Toussaint et al. 2005).

In pigs the typical APP kinetic shows a distinct increase of the main positive

APPs haptoglobin, pig-MAP, SAA and CRP with values up to 10 - 100 times high-

er than baseline during 2 and 3 days after infection (Heegaard, Klausen et al.

1998, Petersen, Nielsen et al. 2004, Pomorska-Mol, Markowska-Daniel et al.

2011). Concurrently the major negative APP ALB, that is synthesised by hepato-

cytes exclusively, decreases at least 25% below baseline (Fleck, Colley et al.

1985, Goyarts and Dänicke 2006, Kullik, Brosig et al. 2013).

Spreading local infections as well as a prolonged exposure to pathogens such

as LPS provoke severe metabolic, endocrine and immunological alterations sys-

temically (Besedovsky and Del Rey 2001, Gruys, Toussaint et al. 2005). Further

the inflammatory process passes from innate to acquired immunity by humoral

and cell-mediated immune response (Delves and Roitt 2000, Delves and Roitt

2000, Parkin and Cohen 2001).

In accordance to severity of systemic modifications, which are mainly mediated

by TNF-α and IL-1 (Hewett, Jean et al. 1993, Hewett and Roth 1993, Cavaillon

and Adib-Conquy 2005), an inflammatory shock occurs when the cellular oxygen

demand outweighs the supply. The ascertain consequences of shock in swine

are characterised by an initial hyperdynamic phase with normal high blood pres-

sure but increased cardiac output and decreased total peripheral vascular re-

sistance. Possibly this phase can be followed by converse hypodynamic condi-

tions with low arterial blood pressure, decreased cardiac output and increased

Background

- 18 -

total peripheral vascular resistance (Schrauwen and Houvenaghel 1985,

Schrauwen, Cox et al. 1988, Holger, Dries et al. 2010, Wyns, Plessers et al.

2015). Based on this, less perfusion and therefore decreased oxygen supply of

tissues and organs induced cytotoxic effects (Dahm, Thorne et al. 1999,

Andersson, Fenhammar et al. 2010) that appears in pigs by increased apoptosis

in liver, spleen and kidney cells as well as in blood, thymus, lymph nodes and

spleen MNC´s (Cirelli, Carey et al. 1995, Norimatsu, Ono et al. 1995, Haendeler,

Messmer et al. 1996, Nakajima, Mikami et al. 2000, Ebdrup, Krog et al. 2008,

Solling, Nygaard et al. 2011). Especially the porcine liver, that is playing an im-

portant role in acute phase response and clearing LPS from organism´s circula-

tion (Hewett and Roth 1993) shows marked histological alterations after LPS ex-

posure. These include patchy dark red coloured surfaces, haemorrhages of dif-

ferent sizes (e.g. petechiae, ecchymoses and sugillations), leukocyte infiltration

(neutrophils and eosinophils), endothelial damage, sinusoidal dilatation and

edema, lipid accumulation, as well as damage of hepatocytes and phagocyting

KUPFFER cells (Cirelli, Carey et al. 1995, Saetre, Hovig et al. 2001, Stanek,

Reinhardt et al. 2012).

In addition to these organic implications further systemic alterations, generally

mediated by cytokines´ action on the hypothalamus and its subordinated HPA

axis (Chrousos 1995, Mackowiak 1998, Annane, Bellissant et al. 2005, Roth and

Blatteis 2014, Wyns, Plessers et al. 2015), are reflected in pigs by clinical symp-

toms (Dänicke, Brosig et al. 2013). Especially under practical conditions, clinical

symptoms are the first and easiest detectable parameters, characterising an ac-

tive immune response to toxins such as LPS. In pigs typical signs for endotox-

aemia are fever, sickness behaviour (e.g. somnolence, lethargy, shivering, ano-

rexia, retching and vomiting), an increase in respiratory rate, reddened conjunc-

tivae as well as injected episcleral vessels occurring in a time-dependent se-

quence, presumably based on the sequential appearance of pro-inflammatory

cytokines. The cytokine cascade is starting with TNF-α, reaching peak concen-

tration 1 h after intoxication, immediately thereafter PGE2 at 1 - 2 h, followed by

IL-6 at 2.5 h and IL-1ß at 3.5 h (Wyns, Plessers et al. 2015). One of the main

symptoms due to a systemic inflammatory response is fever, even though the

Background

- 19 -

mechanisms of fever production have not been sufficiently clarified yet. Howev-

er, PGE2 is established as key mediator, inducing an increase in body tempera-

ture by binding on thermoregulatory neurons in the hypothalamus. Additionally

pyrogenic cytokines such as TNF-α, IL-1ß and IL-6 are accepted to play a major

role in induction of such febrile responses (Fraifeld and Kaplanski 1998,

Mackowiak 1998, Roth and Blatteis 2014, Wyns, Plessers et al. 2015). Other

marked symptoms such as cyanosis, hyperaemic conjunctivae and injected

episcleral vessels are based on mentioned cellular or rather cytokine interactions

that lead to vascular endothelial damage and permeability changes.

Besides these observable alterations, sickness behaviour also provoke a

dysregulation of metabolic homeostasis during early inflammation (Elsasser,

Kahl et al. 2000, Humphrey and Klasing 2004) by the reduction of feed intake

and thus the restriction of dietary input of proteins. Regardless, the uptake of

amino acid by the liver increases severely and in lymphoid organs moderately

(Humphrey and Klasing 2004) to ensure the greater need for an adequate

amount of energy realising for instance the fever response, synthesis of protec-

tive factors (e.g. complement, APP, cytokines, antibodies) and leukocyte prolifer-

ation or certain metabolic pathways (Baracos, Whitmore et al. 1987, Dahn,

Mitchell et al. 1995, Biolo, Toigo et al. 1997, Chiolero, Revelly et al. 1997). This

results in endogenous TNF-α regulated protein breakdown, primarily occurring in

skeletal muscles (Rosenblatt, Clowes et al. 1983, Baracos, Whitmore et al.

1987, Sax, Talamini et al. 1988, Chiolero, Revelly et al. 1997, Cooney, Kimball

et al. 1997, Gruys, Toussaint et al. 2005) Additionally the brain also increases its

uptake of the amino acid TRP for its increased synthesis of serotonin, that in

turn, is described to be responsible for anorectic effects (Schröcksnadel,

Wirleitner et al. 2006, O'Connor, Lawson et al. 2009).

3. Interactions between DON and LPS

Both toxins, DON as a feed contaminant, and LPS as part of commensal bacte-

ria, mainly enter the blood circulation via the portal vein by absorption from the

GIT just as nutrients (van Deventer, Buller et al. 1990, Goyarts and Dänicke

2006, Andreasen, Krabbe et al. 2008, Döll and Dänicke 2011). Thus they firstly

Background

- 20 -

achieved the liver, which is not only the central metabolic organ for key nutrients

such as carbohydrates and proteins but also facilitating systemic inflammatory

reactions such as processing and eliminating toxic agents via hepatic KUPFFER

cells contributing to local and systemic effects (Maresca 2013). Whereby, sub-

stances entering the liver in the first pass (via portal route) are to be expected to

trigger a different response in contrast to agents in systemic circulation that enter

the liver only in a second pass.

Based on the common modes of action of DON and LPS an interaction between

both is generally assumed and presented in several studies.

Rodent research data showed that LPS priming potentiates the DON induced

increase in IL-6 and TNF-α mRNA expression in murine cell lines (Pestka and

Zhou 2006). While in vivo results indicate that LPS priming sensitises the organ-

ism to DON, which is shown in a shortened onset time and simultaneously in-

creasing magnitude and duration levels of IL-6, TNF-α serum proteins and splen-

ic mRNA (Islam and Pestka 2006). Also synergistic and additive effects were

found in mice, concurrently exposed to both toxins. These data showed an in-

crease in concentrations of IL-6, TNF-α and splenic mRNA level (Zhou, Harkema

et al. 1999) as well as increased apoptosis and lymphoid atrophy in the thymus,

Peyer´s patches, bone marrow, spleen and liver (Zhou, Harkema et al. 2000,

Islam and Pestka 2003).

Studies in pigs showed similar results to rodent data. In porcine pulmonary alve-

olar macrophages and KUPFFER cell enriched hepatocytes, DON and LPS syner-

gistically induced a marked higher increase of TNF-α protein levels and mRNA

expression compared to single toxin exposure (Döll, Schrickx et al. 2009, Döll,

Schrickx et al. 2009). These interactive effects were also found in swine infused

with both toxins synchronously. Beside a significantly attenuated TNF-α peak

(Dänicke 2013) animals also showed significantly elevated bilirubin levels com-

pared to sole toxin administration (Stanek, Reinhardt et al. 2012). Additionally, it

was also reported that DON-feeding combined with an ensuing PAMP administra-

tion modifies the APR. Relating thereto published findings show an attenuated

immune response to sheep erythrocytes (Rotter, Thompson et al. 1994) as well

as decreased cytokine and immunoglobulin expressions and moderate impacts

Background

- 21 -

on lymphocyte proliferation after an injection of ovalbumin (Grenier, Loureiro-

Bracarense et al. 2011). Both stimuli primarily encourage the adaptive immune

system and generally cause a secondary antibody response that does not coin-

cide with the stimulation of the innate immune system and its inclusion of APR.

Additionally pigs, stimulated with LPS, after feeding a DON-contaminated diet

showed an attenuation of hepatic histopathological lesions (Stanek, Reinhardt et

al. 2012).

Albeit interactive effects between DON and LPS are scarcely reported for clinic

and haematology so far, but those few reports in pigs showed no significant im-

pact of combined toxin exposure with respect to clinical symptoms, white blood

cell counts and cytokine expression (Dänicke, Brosig et al. 2013).

Scope of thesis

- 22 -

Scope of thesis

The background outlines a large pool of knowledge about the modes of action of

both toxins, DON and LPS. However, data about their metabolic and immunologi-

cal interactions in animals are subject to large variations depending on examined

species, toxin concentration and the way of exposure. Pigs are known as the

most sensitive species to DON, but in this context, the knowledge about the im-

portance of the liver as central organ of metabolism, detoxification and immune

response is still limited in swine.

For that reason, the aim of the present thesis was to elucidate potential hepatic

modifications of the APR as well as its associated clinical signs, inflammatory

markers and their relationships in pigs exposed to a chronic dietary DON com-

bined with an acute pre- or post-hepatic LPS-induced endotoxaemia.

In this thesis the following hypotheses have to be verified:

1. The liver differently mediates the APR by modifications of its metabolising and

detoxifying properties against an acute LPS-intoxication.

2. LPS that passes the liver first (pre-hepatic infusion) is proportionally removed

before reaching the blood circulation, thus clinical signs are less pronounced

compared to direct systemic intoxication (post-hepatic infusion).

3. Clinically inconspicuous pigs (CON-/DON-fed, NaCl-infused) exhibit a con-

sistent relationship between body core and rectal temperature measurement

as well as their linkage to inflammatory blood parameters.

4. In pathophysiological conditions (CON-/DON-fed, LPS-infused) the liver-

depending alterations in APR are reflected in an inconsistent relationship be-

tween distinct body temperature measurements as well as their linkage to in-

flammatory markers.

For this purpose we conducted a study with 44, ten weeks old barrows (German

Landrace with an initial mean BW of 25.8 ± 3.7kg), that were divided in two feed-

ing groups over a period of four weeks. One group was fed with an almost DON-

free diet (CON) while the other group received a naturally DON-contaminated diet

(DON), containing 4.59 mg DON/kg feed. To exclude nutrient and energy effects

Scope of thesis

- 23 -

as well as associated alterations in haematological and serum parameters, pigs

were fed restrictively (2 x 700g/pig and d). At 48 h before the LPS-challenge pigs

were surgically equipped with a multi-catheter system that facilitates the creation

of different conditions focusing on hepatic functions by pre- (V. splenica) or post-

hepatic (V. jugularis ext.) NaCl - or LPS-infusion (0.9% NaCl or 7.5µg LPS/kg BW

for 60 min) combined with simultaneous blood sampling (V. portae hepatis, V.

jugularis int.). Including the two levels of dietary (DON exposure) and both sites

of (NaCl - or LPS-) infusions, the actual study comprised six experimental

groups: CON_ CONjug.-CONpor., CON_CONjug.-LPSpor., CON_LPSjug.-CONpor.,

DON_CONjug.-CONpor., DON_CONjug.-LPSpor., DON_LPSjug.-CONpor..

In consideration of the initially mentioned aim of the study, two scientific articles

have been published.

Figure 5: The first publication (Paper I) observed the impact of sole DON-feeding, pre- and post-

hepatic LPS-infusion as well as their combinations on clinical signs, body core temperature, WBC

counts and TNF-α, while the second publication (Paper II) involves KYN, TRP, KYN-TRP ratio and

rectal temperature measurement as additional inflammation indicators. Furthermore, these pa-

rameters and the previously analysed data (Paper I) were considered in more detail by relating

them to each other.

Paper I

- 24 -

PAPER I

Does dietary deoxynivalenol modulates the acute

phase reaction in endotoxaemic pigs?

- Lessons from clinical signs, white blood cell counts

and TNF-alpha -

Tanja Tesch1, Erik Bannert1, Jeannette Kluess1, Jana Frahm1, Susanne Kers-

ten1, Gerhard Breves2, Lydia Renner3, Stefan Kahlert3, Hermann-Josef Rothköt-

ter3 and Sven Dänicke1

1 Institute of Animal Nutrition, Friedrich-Loeffler Institute (FLI), Federal Research Insti-

tute of Animal Health, Braunschweig, Germany

2 Institute for Physiology, University of Veterinary Medicine, Foundation, Hannover,

Germany

3 Institute of Anatomy, Otto von Guericke University Magdeburg, Magdeburg,

Germany

Toxins

2016

Volume 8/3

DOI:10.3390/toxins8010003

Paper I

- 25 -

Abstract: We studied the interaction between deoxynivalenol (DON)-feeding

and a subsequent pre- and post-hepatic immune stimulus with the hypothesis

that the liver differently mediates the acute phase reaction (APR) in pigs. Bar-

rows (n = 44) were divided into a DON-(4.59 mg DON/kg feed) and a control-diet

group, surgically equipped with permanent catheters pre- (V. portae hepatis) and

post-hepatic (V. jugularis interna) and infused either with 0.9% NaCl or LPS (7.5

µg/kg BW). Thus, combination of diet (CON vs. DON) and infusion (CON vs.

LPS, jugular vs. portal) created six groups: CON_CONjug.-CONpor., CON_CONjug.-

LPSpor., CON_LPSjug.-CONpor., DON_CONjug.-CONpor., DON_CONjug.-LPSpor.,

DON_LPSjug.-CONpor.. Blood samples were taken at −30, 15, 30, 45, 60, 75, 90,

120, 150, 180 min relative to infusion and analysed for leukocytes and TNF-

Paper I

- 26 -

alpha. Concurrently, clinical signs were scored and body temperature measured

during the same period. LPS as such induced a dramatic rise in TNF-alpha (p <

0.001), hyperthermia (p < 0.01), and severe leukopenia (p < 0.001). In CON-fed

pigs, an earlier return to physiological base levels was observed for the clinical

complex, starting at 120 min post infusionem (p < 0.05) and persisting until 180

min. DON_LPSjug.-CONpor. resulted in a lower temperature rise (p = 0.08) com-

pared to CON_LPSjug.-CONpor.. In conclusion, APR resulting from a post-hepatic

immune stimulus was altered by chronic DON-feeding.

Keywords: pig; deoxynivalenol; lipopolysaccharide; liver; leukocytes; clinical symptoms;

tumor necrosis factor alpha; body core temperature

1. Introduction

The B-trichothecene deoxynivalenol (DON) is a mycotoxin mainly produced by

the plant pathogens Fusarium graminearum and F. culmorum. It is often detect-

ed in cereal grains, especially wheat and maize. These are major portions of

farm animal’s diets, which are therefore frequently contaminated with toxicologi-

cally relevant levels. Pigs are known as the most DON-sensitive species [1,2]

and symptoms such as vomiting, inappetence, and reduced weight gain are of-

ten related to DON-contamination of the pig feed. Moreover, DON effects are

dependent on dosing regime and frequency of exposure and can be immuno-

suppressive at acute high-doses, e.g., reflected by rapid increase of leukocyte

apoptosis or immunostimulatory at low-doses, e.g., resulting in an increased ex-

pression of cytokines. It is conceivable that a large proportion of animals in

swine production might encounter chronic dietary DON exposure during their

lifetime and, given the infectious pressure in the production site, might also be

co-exposed to a systemic infection such as Salmonellosis, Campylobacter, or E.

coli infections [3,4]. Because DON has been reported to modify the organism’s

immune response, this poses the question whether pigs pre-exposed to dietary

mycotoxins react with an altered response, e.g., in the clinical progression of an

occurring systemic infection and thus the ability of the organism to deal with this

disease. Investigating such a potential interaction of mycotoxin exposure and

systemic infections one needs to use an established infection or inflammation

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animal model enabling the researcher to discern the known effects of the infec-

tion from the to-be-investigated additional mycotoxin impact. The endotoxin

(LPS) is a well-established model substance, challenging the immune system

and thus simulating an inflammatory state usually present in a systemic infec-

tion. LPS is a component of the outer cell membrane of gram-negative bacteria

and consists of a hydrophobic domain as the source of toxicity (lipid A), a core

region (oligosaccharides), and a hydrophilic O-specific chain responsible for

most antigenic properties (polysaccharide) [5].

Data from rodent studies reported synergistic effects between DON and LPS

with respect to the acute phase reaction (APR) in particular the induction of pro-

inflammatory cytokine expression [6,7]. Furthermore, LPS priming potentiated

not only the pro-inflammatory response to DON exposure but also the toxicity of

both [8,9].

In the challenge model, LPS has a high stimulatory potential for the innate and

acquired immune system due to the interaction with different types of leukocytes

initiating, amongst other things, the biosynthesis of various mediators of inflam-

mation, such as TNF-alpha (TNF-α). This is the first pro-inflammatory cytokine in

the cytokine cascade playing a major role in fever induction as well as in the oc-

currence of related clinical signs as the first and easiest observable changes in

vivo. Thus, the response of the immune system to LPS in animals pre-exposed

to DON might be reflected in altered differential blood cell counts and clinical

manifestation of an APR resulting from increased levels of TNF-α and other in-

flammatory mediators [5].

Both DON, as a feed contaminant, and LPS, as part of commensal bacteria,

mainly enter the organism via the gastrointestinal tract (GIT) where they can be

absorbed into the blood stream [1,10–12] The GIT blood flow is drained into the

portal vein (portal drained viscera, PDV) and all absorbed substances such as

nutrients or even toxins enter the liver via this route. The liver is not only the cen-

tral metabolic organ for the key nutrients such as carbohydrates and proteins,

but also processes and eliminates toxic agents, e.g., DON is glucuronidated in

the liver [13]. Furthermore, systemic inflammatory reactions are also facilitated

via hepatic KUPFFER cells contributing to local and systemic effects. Therefore,

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substances absorbed from the GIT and entering the liver in a so-called first pass

are likely to elicit a different response compared to their counterparts in systemic

circulation, gaining entry to the liver only in a second pass. Although this is a

crucial point, there are only scarce data discerning immune stimulation and its

response pre- and post-hepatically, in particular regarding myco- and endotoxin

exposure. Thus, we wanted to test the hypothesis that the liver differently medi-

ates the systemic inflammatory response to an acute LPS-stimulus in chronically

DON-fed pigs. Therefore, we employed a pig model enabling portal (pre-hepatic)

and peripheral (post-hepatic) access for assessing the liver’s contribution to the

pig’s response to myco- and endotoxin exposure.

2. Results

2.1. Clinical Score

Ten clinical symptoms characterizing an inflammatory reaction were observed in

a time kinetic manner and scored for their presence and severity for all experi-

mental groups. Data for all symptoms and all times tested were subsumed in a

cumulative clinical score (CCS) for each experimental group representing a clini-

cal complex. All LPS exposed pigs had a significantly higher CCS compared to

control-infused groups. Viewing the four LPS-exposed group revealed that por-

tal-infused groups showed numerically lower CCS compared to their jugular-

infused counterparts, but this could not be statistically verified. DON-feeding

alone (DON_CONjug.-CONpor.) did not alter clinical symptoms and combined

with LPS did not alter the LPS-reaction. At least 5 out of 10 detected individual

clinical symptoms indicative for an acute endotoxaemia were represented in

LPS-infused groups, while both control infusion groups displayed only sporadi-

cally adverse symptoms (Figure 1).

The most pronounced symptoms observed in all LPS-pigs were tremor, cyano-

sis, injected episcleral vessels and hyperaemic conjunctivae showing a coherent

time kinetic (Figure 2a,b), whereas nystagmus, teeth gnashing, and dermogra-

phism were sporadic symptoms that occurred only in a few LPS-infused animals

(n = 5 out of 28 LPS-pigs). Although labored respiration, increased respiratory

rate, retching, and vomiting were also mostly detected in LPS-infused pigs,

these symptoms did not show a clear sequence of time.

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Figure 1. The cumulative clinical score (CCS) represents the entire clinical complex induced by

the six experimental treatments, i.e., chronic feeding strategy (CON vs. DON) and acute chal-

lenge situation (CON vs. LPS). Each CCS bar consists of maximum 10 clinical symptoms scored

at 13 points in time over the entire observation period of 210 min for each group. LPS-infused

groups had a significant higher CCS and considerably more symptoms than control-infused

groups, irrespective of dietary treatment. Data represent LSmeans (PSEM ± 8.6) and statistical

main effects were distributed as follows: p group < 0.001. Bars with no common superscripts (a,b)

are significantly different (p < 0.001).

The four key symptoms (Figure 2a,b) had a uniform sequence starting between

15 and 30 min post infusionem (p.i.) with tremor and cyanosis followed by inject-

ed episcleral vessels and hyperemic conjunctivae in LPS-groups. Control-

infused groups showed no adverse clinical symptoms and thus were not includ-

ed in Figure 2a,b. We calculated also the CCS for each point in time (identical to

Figure 1) in order to discriminate the influence of DON and LPS on the course of

the clinical symptoms (Figure 2c). Here we observed that the site of LPS-

infusion, i.e., pre- or post-hepatically, had a stronger impact on the clinical pro-

gression as compared to the mycotoxin influence. Within CON-fed groups portal

infusion caused a significantly earlier return (120 min p.i., p = 0.05) to physiolog-

ical levels compared to jugular infusion and this effect persisted until 180 min

p.i.. In DON-fed pigs, jugular infusion showed a significantly higher score in clini-

cal symptoms at 15 min p.i. (p = 0.04) compared to their portal infused counter-

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parts, but no other differences could be verified.

Figure 2. LPS caused a sequential series of four key symptoms and the kinetic of those individ-

ual clinical signs is depicted in the two upper graphs for each dietary treatment. (a) showing

CON-fed and (b) DON-fed groups. Tremor reached its highest level already between 30 and 45

min while the other symptoms showed there maximum degree between 75 and 105 min. There-

after, from 120 min onwards, symptoms declined slowly to the base level. Data represent

LSmeans (PSEM Tremor ± 0.07, PSEM injected episcleral vessels/cyanosis ± 0.15, PSEM hyperaemic conjunctivae ±

0.11) and statistical main effects were distributed as follows: cyanosis p group < 0.001, p time <

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0.001, p group × time < 0.01, tremor/hyperaemic conjunctivae/injected episcleral vessels p group <

0.001, p time < 0.001, p group × time < 0.001. The cumulative clinical score (CCS) for each point in

time is presented in (c) and the statistical difference (p-value) between jugular and portal infusion

within each feeding group is provided. Data represent LSmeans and statistical main effects were

distributed as follows: p group < 0.001, p time < 0.001, p group × time < 0.001.

2.2. Body Temperature

Body temperature was measured every 5 min with an intra-abdominal tempera-

ture logger (body core temperature). Compared to control-infused animals show-

ing no increase in temperature during the entire observation period, LPS-infusion

induced a significant hyperthermia with an increase of ~1.5 °C, starting from 30

min p.i., irrespective of infusion site or diet. Additionally, a marked diet effect was

found in jugular-infused LPS-pigs: DON-fed animals (p = 0.08) exhibited consist-

ently lower temperature (~0.5 °C) as compared to their LPS-infused, CON-fed

counterparts (Figure 3). When comparing the two feeding-groups at each point

in time, t-test revealed that, during the period 30 to 115 min p.i., DON-fed pigs

displayed significantly lower temperatures (p < 0.05).

Figure 3. Body core temperature measurement (in 5 min intervals) for all experimental groups.

All LPS-infused groups increased significantly and showed a hyperthermia in contrast to control-

infused groups. DON-feeding had also a marked effect in LPS-infused animals and showed con-

sistently lower temperature (~0.5 °C) as compared to their control-fed counterparts. Data repre-

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sent LSmeans (PSEM ± 0.02) and statistical main effects were distributed as follows: p group <

0.01, p time < 0.001, p group × time< 0.001.

2.3. White Blood Cell Counts

White blood cell counts were analysed in arterial and pre- and post-hepatic ve-

nous blood samples, whereby no statistical differences were found between

sampling sites (Table 1). At all sampling locations a severe leukopenia (p <

0.001) was detected in response to LPS-infusion, with counts falling to a mini-

mum of 2.2 × 103/µL, irrespective of infusion site and diet, starting at 30 min p.i..

However, group DON_LPSjug.-CONpor. exhibited an even earlier leukopenia, al-

ready significantly present at 15 min compared to −30 min (p < 0.05), whereas

the other LPS-infused groups showed a significant decrease at 30 min com-

pared to −30 min (p < 0.05). Within the study period no return of leukocyte

counts to base levels was observed in LPS-groups.

Table 1. Time kinetics of total leukocyte counts (103/µL). Physiological range 8 - 16 ×10

3/µL [14].

Data represent LSmeans (PSEM ± 0.7) and statistical main effects were distributed as followed:

p group < 0.001, p sampling site = 0.02, p time < 0.001, p group × sampling site × time < 0.001. Values with no

common superscripts (a,b) are significantly different within columns (p < 0.05).

Prior to the infusion regime at −30 min, DON-fed pigs showed significantly (p =

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0.04) higher leukocyte counts than CON-feed animals (16.7 vs. 14.6 × 103/µL).

This dietary effect was detectable for control-infused groups throughout the

study period, with leukocyte counts in DON-fed animals showing consistently

higher values (~2 × 103/µL) as compared to CON-group.

Figure 4. Kinetics of segmented neutrophil (a) and lymphocyte (b) counts in all experimental

groups, with portal data exemplary for all sampling sites. LPS caused a severe neutrophil de-

crease starting at 15 min p.i.. Data represent LSmeans (SEM ± 0.3) and statistical main effects

were distributed as followed: p group < 0.001, p sampling site = 0.03, p time < 0.001, p group × sampling site ×

time < 0.001. LPS caused a more slowly decrease in lymphocytes, also starting at 15 min p.i..

Data represent LSmeans (SEM ± 0.3) and statistical main effects were distributed as followed:

p group < 0.001, p sampling site < 0.001, p time < 0.001, p group × sampling site × time < 0.001.

The decrease of total leukocytes was confirmed in white blood cell differentiation

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with a severe neutropenia and lymphopenia in LPS-infused groups while leuko-

cyte types in both control groups were stable in the physiological range.

Segmented neutrophils (× 103/µL or %) decreased in LPS infused groups, start-

ing 15 min p.i. and falling abruptly below physiological range (4 - 9.6 × 103/µL or

50% - 60%; [14]) at 45 min, irrespective of infusion site or diet (Figure 4).

In comparison, lymphocyte percentage increased significantly at 30 min p.i.

while total counts decreased de facto due to the severe leukopenia addressed

above (Table 1) gradually below the physiological values (2.8 - 8 × 103/µL or

35% - 50%; [14]). Monocytes (× 103/µL) and eosinophils (× 103/µL) decreased in

LPS-infused groups significantly starting at 45 min p.i. while basophil total counts

showed only slight variations, contributing to significant group, time and group ×

sampling site × time interaction. No change at all was found in banded neutro-

phils at all, representing immature neutrophils usually invading from bone mar-

row to replace the mature segmented neutrophils in blood.

2.4. TNF-Alpha

TNF-α, an early pro-inflammatory cytokine, was analysed in pre- and post-

hepatic venous blood samples, whereby no differences were found between

sampling sites. Control-infused animals showed values < 1 ng/mL during the

entire experimental period, comparable to the respective base-level at -30 min.

In LPS-infused groups a severe, sudden increase of TNF-α started at 30 min p.i.

and peaked at 60 min (~140 ng/mL), irrespective of infusion site and diet.

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Figure 5. Kinetics of TNF-alpha measured in (a) portal and (b) jugular blood samples. Control-

infused groups showed consistently unchanged TNF-alpha values of 0.4 ng/mL ± 0.03 during the

entire experimental period. LPS-infusion elicited a strong increase of the cytokine with peak-

values at 60 min, irrespective of dietary treatment or site of infusion. Data represent LSmeans

(PSEM±) and statistical main effects were distributed as followed: p group < 0.001, p sampling site =

0.46, p time < 0.001, p group × sampling site × time < 0.001.

Subsequently, TNF-α decreased to values below half the peak value until 120

min and returned almost back to baseline at 180 min p.i. (Figure 5).

3. Discussion

Earlier experiments investigating the interactions between oral DON exposure

and intravenous LPS administration in pigs were limited to examinations at the

systemic level only and thus neglected the crucial role of the liver in the clear-

ance of absorbed DON and LPS [15]. Therefore, we specifically addressed the

role of the liver by infusing LPS into the portal vein, mimicking a gastro-intestinal

originated flooding of the liver by LPS. This increased hepatic LPS-influx has

been proposed to be mediated by a DON-induced impairment of the intestinal

barrier [16-18]. Enabling LPS administration via the portal or systemic route in

pigs exposed orally to either DON or CON diet, we wanted to test the hypothesis

that the liver differently mediates the systemic inflammatory response as mani-

fested by clinical signs as well as alterations in the plasma concentration of pro-

inflammatory cytokines and white blood cell counts.

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In our pigs we found typical signs for a systemic inflammatory response charac-

terized by an increase in respiratory rate, reddened conjunctivae, injected epis-

cleral vessels, shivering, retching and vomiting, as well as a marked fever re-

sponse associated with a severe leukopenia based on neutropenia and lympho-

penia, in all LPS-infused animals, without regard to any impact by DON-feeding

or infusion site. Most of these symptoms we detected had a time-dependent uni-

form sequence starting 15 min p.i. and reaching their highest scores from 75 min

p.i. onwards. Similar LPS-induced alterations were also found in previous stud-

ies with endotoxemic pigs in a comparable experimental setup [19] and in other

large animal and rodent septic models [20,21].

The time-dependent sequence of symptoms is presumably depending on the

sequential appearance of pro-inflammatory cytokines, released from immuno-

competent cells [22,23]. In our LPS-infused pigs TNF-α reached its peak con-

centration at 1 h p.i., which was associated with a simultaneous fever response

as one of the main symptoms of a systemic inflammatory response. Data from

studies with a similar experimental setup detected analogical clinical signs and

these symptoms coincided with a rise in systemic plasma concentration of pro-

inflammatory mediators (TNF-α, IL-6), clearly induced by LPS [15,23]. Based on

the observed alterations, mentioned above, as well as the two different sites of

infusion, we considered our endotoxemic porcine model as well-suited for inves-

tigating the systemic and hepatic effects of a LPS-induced APR. However, in the

present study we failed to detect any differences in TNF-α kinetics caused by the

pre- or post-hepatic entry of LPS into the liver. This result suggests that the

amount of LPS entering the organism at any site was sufficient to mount a max-

imum inflammatory response. As LPS-infusion via the portal route likely resulted

in a much larger total hepatic LPS load as compared to the systemic route of

administration, it might be concluded that the stimulation of extrahepatic TNF-α

generating cells resulted in comparable TNF-α kinetics and subsequent se-

quences of clinical signs.

In addition to these LPS-effects, we found an impact of chronic enteral DON-

exposure. Sole DON-feeding induced a significant increase in total leukocyte

counts close to the upper physiological range. This impact of DON is also re-

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ported after a 28 day exposure of oral low DON dosage (0.75 - 3 mg/kg) in

young swine [24] and. after an i.v. DON-injection (1 mg/kg BW) in miniature pigs

[25]. Their data showed a significant increase of leukocytes and neutrophils due

to DON whereas other studies in growing pigs exposed to an oral low DON dos-

age (0.28 - 3 mg/kg feed) for 32 days [26-28] or to a 1 h DON-infusion (0.1

mg/kg/BW) [19] found no impact of DON on white blood cell counts. These

DON-effects on the leukogram have been ascribed to co-contaminations with

other mycotoxins and to changes in feeding behavior and thus an altered nutri-

tional status by various authors [26,29]. In order to prevent changes in feeding

behavior, pigs in our study were fed restrictively and therefore the detected im-

pact on leukocyte counts can be attributed to the immunological response of the

organism to DON.

Moreover, it is reported that DON shows interactive effects with a further im-

mune stimulus, such as LPS, resulting in a modified APR. Although our data

could not confirm this interaction on TNF-α, previous studies have reported on a

significant attenuation of the TNF-α peak in pigs infused with the combination of

DON (0.1 mg/kg BW) and LPS (7.5 µg/kg BW) compared to both toxins alone

[15]. However, we found significantly interactive effects between DON and LPS

on body core temperature and clinical symptoms but those seemed to be de-

pendent on the site of infusion. Similar interactive effects of both toxins, irrespec-

tive of infusion site, were detected in different studies in mice treated with a sin-

gle oral DON dose (25 mg/kg BW dissolved in 0.25 mL) and an intraperitoneal

LPS-injection (0.5 mg/kg/BW dissolved in 0.25 mL) [30,31]. The co-exposure of

LPS and the mycotoxin showed, amongst other things, an induction of apoptosis

of immune cells in liver, spleen, thymus, and Peyer´s patches with a synergistic

impact of both toxins on thymus and spleen [30]. Furthermore, other porcine

studies showed an attenuation in hepatic histopathology when pigs were fed a

DON-contaminated diet (3.1 mg/kg feed, 37 d) and received a subsequent LPS-

infusion (7.5 µg/kg BW) [32]. In simultaneously DON and LPS infused pigs fed

with a control diet both toxins strongly interacted with each other and elevated

bilirubin levels significantly in contrast to the sole toxin administration [32]. This

contrast was also verified in our experimental pigs, demonstrating a dramatic

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impact on development of lactic acidosis in DON-fed pigs receiving peripheral

LPS [33]. Other comparable interactions for clinic and hematology are scarcely

reported in pigs so far and those few reports show no significant impact of DON

combined with LPS on clinical symptoms, white blood cell counts, and cytokine

expression in pigs [15,34].

Furthermore, only few experimental data elucidating the liver’s involvement in

this context have been published yet. Our data showed various alterations

caused by infusion-site of LPS and its combination with diet. CON-fed pigs, after

LPS-portal infusion, returned even more rapidly to physiological clinical levels

than their post-hepatic infused counterparts. This is probably explained by the

fact that a portal-infused (pre-hepatic) immune stimulus such as LPS and sub-

stances absorbed from the GIT such as DON have to pass the liver (first-pass)

before reaching systemic blood circulation. Thus, those pathogenic agents could

proportionally be removed by the liver to reduce the pressure for the rest of the

organism, while a jugular-infused (post-hepatic) stimulus spreads through the

whole blood system before passing the liver (second-pass). The latter might also

explain that in our study a chronic DON exposure shortened the response time

of leukocytes and accelerated the occurrence of associated clinical signs at 15

min when pigs were post-hepatically LPS-infused. Additionally, these pigs

showed a significantly lower elevation of body core temperature compared to

their CON-fed counterpart. This interaction between DON and LPS with respect

to body core temperature also tended to be seen in portal-infused groups. To the

best of our knowledge, this is the first time that such an interaction of DON-

feeding and peripheral LPS-stimulus on body core temperature is reported in

pigs. However, a previous study in mice could demonstrate an impact of DON

per os on body temperature and central inflammation [35]. Therein wild-type

mice, receiving an oral bolus of DON at different concentrations, exhibited a

dose-dependent, transient decrease in body temperature, lasting at least 6 h. At

the level of the central nervous system, mice showed, inter alia, significantly up-

regulated TNF-α and COX-2 transcripts in hypothalamus and dorsal vagal com-

plex. Centrally upregulated TNF-α is proposed to act as an endogenous cryo-

gen, lowering the body temperature in response to DON that is indeed observed

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in these mice. Consequently, we hypothesize that our DON-fed pigs experienced

a similar central nervous effect, thus effectively resulting in the observed lower

elevation in body temperature in response to a subsequent LPS-treatment com-

pared to their control-fed counterparts.

Whether this DON impact on body temperature can be appraised as positive or

negative for the pig’s organism remains to be elucidated. Such an increase in

body temperature can positively support the inflammatory response in counter-

acting live pathogens, but is also considered as detrimental for the body’s tissue

when passing an upper threshold. Therefore, a successful elevation in body

temperature should be regulated on the lowest possible level. One could argue

that the organism is more capable in successfully counteracting or even eliminat-

ing LPS-mediated effects when being exposed to prior DON-feeding.

In conclusion, the present study indicated that chronic oral DON-exposure

modulates the metabolising and detoxifying properties of the porcine liver

against acute LPS stimulus. This confirms our hypothesis that the liver differently

mediates the systemic inflammatory response, especially in leukocytes, body

core temperature, and clinical signs.

4. Experimental Section

Experiment and procedures were conducted according to the European Com-

munity regulations concerning the protection of experimental animals and the

guidelines of the German Animal Welfare Act and were approved by the ethical

committee of the Lower Saxony State Office for Consumer Protection and Food

Safety (file number 33.4-42502-04-13/1274).

4.1. Experimental Design

To investigate the effects of dietary DON combined with an intravenous LPS

stimulus on acute phase reaction in pigs, a total of six experimental groups were

tested (Figure 6).

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Figure 6. The feeding trial took place over a period of four weeks and the general experimental

setup, including experimental groups is depicted in the upper panel. On day 27, pigs were surgi-

cally equipped with arterial and venous catheters followed by a recovery day. After the morning

feeding (6:45 - 7:00) on day 29, animals were exposed to acute intravenous treatments for 1 h

thereby creating six experimental groups in total. (lower panel) Over a period of 210 min, starting

30 min before infusion until 180 min p.i. blood samples were taken and clinical signs were ob-

served.

4.2. Animals and Diets

The study was accomplished using a total of 44 barrows (German Landrace,

Mariensee, Germany) with an initial mean body weight (BW) of 25.8 ± 3.7 kg.

Animals were divided in two feeding-groups: one group was chronically exposed

to a diet incorporating maize naturally contaminated with DON and the other

group received the same diet with non-contaminated maize (Table 2). DON was

measured in both compound diets using an HPLC-method as reported

earlier [36] and values are provided in Table 2. During the feeding trial pigs were

kept separately in floor pens and fed restrictively twice daily in equal quantity (2

× 700 g/animal and day). Thus, each pig received 6.43 mg DON per day. Ani-

mals were accustomed to balance cages and fasted the night before surgery.

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Table 2. Composition of experimental diets (based on dry matter content of 88%).

4.3. Surgery

Following an initiate intramuscular application of tiletamin-zolazepam (4.4 mg/kg

BW, Zoletil ® 100 mg/mL, Virbac AG, Glattbrugg, Switzerland), atropine (0.04

mg/kg BW, Atropinum sulfuricum 0.5 mg Eifelfango ®, Eifelfango, Bad Neu-

enahr-Ahrweiler, Germany) and carprofen (4mg/kg BW, Rimadyl ® 50 mg/mL,

Pfizer, Münster, Germany) surgery took place under sterile conditions and gen-

eral inhalation anasthesia maintained with isofluran (Isofluran CP, CP-Pharma,

Burgdorf, Germany).

Pigs were equipped with five catheters (Silastic ® Medical Grade Tubing, 1.57

mm ID × 3.18 mm OD, Dow Corning, Midland, MI, USA) covering pre- and post-

hepatic area for contemporaneous blood sampling and infusions. Beginning with

a median laparotomy first catheter was put in via an access by a jejunal vein

through the cranial mesenteric vein into the Vena portae hepatis for sampling

from the portal drained viscera (PDV). The second catheter goes via an access

by a vein of the great curvature to the Vena splenica for applications. After being

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fixed catheters were exteriorized subcutaneously to the left flank and attached

with catheter clamps with fastener (Arrow ®, Arrow International Inc., Reading,

PD, USA). Additionally, a temperature logger (Thermochron i-Button, Maxim in-

tegrated™, San Jose, CA, USA) was fixed in the abdominal cavity and subse-

quently abdominal incision was closed continuously in two layers. Thereafter, the

left jugular grove was opened to insert a catheter each into the Vena jugularis

externa for applications, Vena jugularis interna for sampling from the post-

hepatic area and Arteria carotis communis for sampling as well. After being fas-

tened, they were tunnelled subcutaneously to the neck and the incision in the

jugular grove was closed with skin suture. Catheters of the portal vein and the

carotid artery were fixated in the vessel using pursestring suture (Premilene ®

5/0, B.Braun Melsungen AG, Melsungen, Germany) whereas remaining cathe-

ters were inserted as previously described in detail (Goyarts and Dänicke, 2006

[10]) and each catheter was provided with three-way-valves for blood sampling

and infusions. Heparinized 0.9% NaCl was used for flushing catheters to main-

tain patency.

4.4. Infusions, Clinic, and Sampling

All infusions were performed using an infusion-pump (IPC-N-4, ISMATEC La-

boratoriumstechnik GmbH, Wertheim, Germany) and infusion-tubes with 2.06

mm inner diameter (PharMed ® Ismaprene, ISMATEC Laboratoriumstechnik

GmbH) at a rate of 32 mL/h per animal and infusion catheter. Infusion was ad-

ministered for 60 min simultaneously into both, V. jugularis externa (post-

hepatic) and V. splenica (pre-hepatic), in each experimental animal. Control

groups were infused with physiological saline in both catheters (CON-CONjug.-

CONpor., DON-CONjug.-CONpor.), while LPS (7.5 µg/kg BW dissolved in 0.9%

NaCl, Escherichia coli O111:B4, Product number L2630, Sigma-Aldrich, St. Lou-

is, MO, USA) was administered either into jugular (CON-LPSjug.-CONpor., DON-

LPSjug.-CONpor.) or portal region (CON-CONjug.-LPSpor., DON-CONjug.-LPSpor.)

and the second catheter was simultaneously infused with physiological saline.

Over a period from -30 min before to 180 min after starting the infusion, different

clinical signs were scored (Table 3) and respiratory rate was counted at -30, 15,

30, 45, 60, 75, 90, 105, 120, 135, 150, 165, and 180 min. In addition, body core

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temperature was measured by an i-Button every 5 min. Furthermore serial blood

samples were collected for leukocyte count, differential white blood cell count

(EDTA Monovette®, Sarstedt AG & Co., Sarstedt, Germany) and TNF-alpha (Li-

Heparin Monovette ®, Sarstedt AG & Co.) at −30, 15, 30, 45, 60, 75, 90, 120,

150, and 180 min. Samples for cytokine analyses were immediately centrifuged

and plasma samples were stored at -20°C for subsequent investigations.

Table 3. Clinical score: Cumulative score calculated from all scores of each symptom over the

whole observation period (10 symptoms × 13 times, maximum score 351) or of each symptom

for every point in time (10 symptoms per time, maximum score 27).

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4.5. Measurements and Analyses

4.5.1. Clinic

At each time clinical symptoms were examined and a score system implemented

to facilitate objective comparisons (Table 3). Scoring was performed by one per-

son only, who was unaware of animal´s treatment at time of clinical observa-

tions. These 10 scored clinical signs were summarized as cumulative clinical

score as a measure of clinical severity, on the one hand comprising all 13 times

and each clinical symptoms with a total score of maximal 351, and on the other

hand comprising all symptoms for each point in time seperatly with a total score

of maximal 27 per time.

4.5.2. White Blood Cell Counts

Serial whole blood samples were anaylyzedimmediately after sampling by an

automatic hematology analyzer (Celltac MEK 6400, Nihon Kohden Europe

GmbH, Rosbach, Germany) for total leukocyte counts.

Additionally, one drop of blood was placed on a glass slide and two blood

smears were prepared of each blood sample in order to differentiate the leuko-

cyte types in duplicates. After air-drying blood smears were stained according to

PAPPENHEIM: Slides were places into MAY-GRÜNWALD solution for fixation and

staining for 3 min and subsequently rinsed in distilled water (pH 7.2) to remove

staining solution. After that, slides were put into GIEMSA solution for counterstain-

ing (15 min) and again rinsed in distilled water (pH 7.2). Dried samples were an-

alysed using brightfield microscopy (Nikon Eclipse E200, Nikon GmbH, Tokyo,

Japan) for morphological differentiation of 100 leukocytes per slide.

4.5.3. TNF-Alpha

TNF-alpha was determined at defined times (-30, 30, 60, 90, 120, 180 min) us-

ing a quantitative ELISA kit (Quantikine® ELISA Porcine TNF-α Immunoassay,

Cat. No. PTA00, R & D System Inc., Minneapolis, MN, USA) according to the

manufacturers manual with a level of detection of 2.8 – 5 pg/mL. Samples were

measured photometrically on a Tecan (Tecan® infinite M200, Tecan Trading

AG, Männedorf, Switzerland) at 450, 540, and 570 nm.

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4.6. Statistics

All statistics were performed by using SAS software (Enterprise Guide, version

6.1, SAS Institute, Cary, NC, USA). Generally, the procedure “MIXED” was used

with group (six experimental groups), sampling site (three locations: A. carotis

comunis, V. jugularis interna, V. portae hepatis), time (10 points in time for blood

samples, 13 points in time for clinical scores) and their interactions included as

fixed factors with a compound symmetry covariance structure for leukocyte

counts and TNF-alpha measurements. Body temperature and clinical signs did

not take the sampling site as a fixed factor into consideration and used an auto-

regressive covariance structure cumulative score.

Effects were regarded to be significant (adjusted Tukey post-hoc test) at likeli-

hood lower or equal to 0.05 while a tendency or trend was assumed for proba-

bilities lower than 0.1 and higher than 0.05.

5. Conclusions

Altogether we were able to show both an effect of sole chronic DON-feeding and

an infusion-site effect of the subsequent LPS-stimulus. Based on our data and

data from literature, we suggest that chronic DON exposure, in combination with

a subsequent immune stimulus, on the one hand overstrains the functional ca-

pacity of the liver as indicated by the earlier and stronger leukopenia and on the

other hand accelerates the innate and adaptive immune response as evidenced

by the more uniform return of clinical symptoms to physiological levels combined

with lower levels of hyperthermia.

Acknowledgments: The authors would like to thank Nicola Mickenautsch, Elenia

Scholz, and Lara Lindner of the Institute of Animal Nutrition, Friedrich-Loeffler-Institute

Braunschweig, Germany, for their excellent technical support in sample analysis and the

“Deutsche Forschungsgemeinschaft” (DFG) for financial support (DA 558/1-4). Moreo-

ver, special thanks to Marc-Alexander Lieboldt for his help in editing the manuscript.

Author Contributions: The project was conceived and designed by Jeannette Kluess,

Jana Frahm, Susanne Kersten, Gerhard Breves, Hermann-Josef Rothkötter and Sven

Dänicke while Tanja Tesch, Erik Bannert, Jeannette Kluess, Jana Frahm, Lydia Renner,

Stefan Kahlert and Sven Dänicke organized and performed the experiments including

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the sample collection. Tanja Tesch, Jeannette Kluess, Jana Frahm and Sven Dänicke

analysed and interpreted the data. The manuscript was wrote by Tanja Tesch whereas

Erik Bannert, Jeannette Kluess, Jana Frahm, Susanne Kersten, Gerhard Breves, Lydia

Renner, Stefan Kahlert, Hermann-Josef Rothkötter and Sven Dänicke were revising the

paper.

Conflicts of Interest: The authors declare no conflict of interest.

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PAPER II

Relationships between body temperatures and

inflammation indicators

under physiological and pathophysiological conditions

in pigs exposed to systemic lipopolysaccharide (LPS)

and dietary deoxynivalenol (DON)

Tanja Tesch1, Erik Bannert1, Jeannette Kluess1, Jana Frahm1, Liane Hüther1,

Susanne Kersten1, Gerhard Breves2, Lydia Renner3, Stefan Kahlert3, Hermann-

Josef Rothkötter3 and Sven Dänicke1

1 Institute of Animal Nutrition, Friedrich-Loeffler Institute (FLI), Federal Research

Institute of Animal Health, Braunschweig, Germany

2 Institute for Physiology, University of Veterinary Medicine, Foundation, Hannover,

Germany

3 Institute of Anatomy, Otto von Guericke University Magdeburg, Magdeburg,

Germany

Journal of Animal Physiology and Animal Nutrition

2017

Volume 1/11

DOI: 10.1111/jpn.12684

Paper II

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Summary: We studied the constancy of the relationship between rectal and in-

traabdominal temperature as well as their linkage to inflammatory markers (leu-

kocyte counts, kynurenine to tryptophan ratio (Kyn-Trp ratio), tumor necrosis fac-

tor alpha (TNF-α) in healthy and in pigs exposed to lipopolysaccharide (LPS)

and/or deoxynivalenol (DON).

Barrows (n = 44) were fed 4 weeks either a DON-contaminated (4.59 mg

DON/kg feed) or a control (CON) diet and equipped with an intraabdominal tem-

perature logger and a multi-catheter system (V.portae hepatis, V.lienalis,

Vv.jugulares) facilitating infusion of 0.9% NaCl (CON) or LPS (7.5 µg/kg BW)

and simultaneous blood sampling. Body temperatures were measured and blood

samples taken every 15 min for leukocyte counts, TNF-α and Kyn-Trp ratio.

Combination of diet and infusion created six groups: CON_CONjug.-CONpor.,

CON_CONjug.-LPSpor., CON_LPSjug.-CONpor., DON_CONjug.-CONpor., DON_

CONjug.-LPSpor., DON_LPSjug.-CONpor..

The relationship between both temperatures was not uniform for all conditions.

Linear regression revealed that an intraabdominal increase per 1°C increase in

rectal temperature was ~25% higher in all LPS-infused pigs compared to NaCl-

infusion, albeit diet and site of LPS-infusion modified the magnitude of this dif-

ference. Inflammatory markers were only strongly present under LPS-influence

and showed a significant relationship with body temperatures. For example, leu-

kocyte counts in clinically inconspicuous animals were only significantly correlat-

ed to core temperature in DON-fed pigs, but in all LPS-infused groups, irrespec-

tive of diet and temperature method. In conclusion, the gradient between body

core and rectal temperature is constant in clinically inconspicuous pigs, but not

under various pathophysiological conditions. In the latter, measurement of in-

flammatory markers seems to be a useful completion.

Keywords: body temperature, physiological, pathophysiological conditions, pig, deoxynivalenol,

lipopolysaccharide

1. Introduction

In healthy conditions living vertebrates are situated in homeostasis whilst keep-

ing their vital functions, such as body core temperature, and different blood pa-

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rameters stable within physiological limits. This balance is maintained against a

large range of intrinsic and extrinsic influences by self-regulatory mechanisms of

the organism ensuring an adequate supply and function of all organs and tis-

sues.

In pathophysiological conditions, such as lipopolysaccharide (LPS) induced

acute endotoxaemia or chronic mycotoxin exposure, the organism initiates an

immune response. In order to eliminate the pathogenic agents rapidly, self-

regulatory mechanisms get impaired causing a dysregulation of homeostasis in

several body functions, e.g. body temperature, white blood cell counts (WBC

counts) and blood gases, (Bannert et al. 2015, Tesch et al. 2015). While acute

internal restoration processes to LPS are accompanied by clinically observable

indicators of an immune response, such as the cardinal symptom fever, immune

responses to chronic mycotoxin deoxynivalenol (DON) often comprises slowly

initiated and less obvious modifications in different parameters, such as leuko-

cyte counts (Tesch et al. 2015).

The endotoxin LPS is a component of the outer cell membrane of gram-negative

bacteria and has a high stimulatory potential for the innate and acquired immune

response characterised by its interaction with different types of leukocytes and

their subsequent biosynthesis of various effector molecules, such as tumor ne-

crosis factor alpha (TNF-α), one of the major fever inducing and amino acid me-

tabolism altering cytokines in mammals (Reid et al. 2001, Spate et al. 2004,

Roth et al. 2014). For instance, TNF-α as well as endotoxin LPS are capable to

initiate the degradation of tryptophan (Trp), an essential amino acid, to its me-

tabolite kynurenine (Kyn) and the neurotransmitter serotonin. The catabolism is

catalysed by the hepatic enzyme tryptophan 2,3-dioxygenase (TDO) and the

enzyme indoleamine 2,3-dioxygenase (IDO), that is strongly upregulated in dif-

ferent types of immune cells and tissues, e.g. lung and spleen, during acute and

chronic immune responses (Moffett et al. 2003, Schrocksnadel et al. 2006). The

activity of IDO can be characterised by the kynurenine to tryptophan ratio (Kyn-

Trp ratio) which correlates with concentrations of immune activation markers,

thus it is an important parameter for the activation of the immune system (Moffett

et al. 2003, Schrocksnadel et al. 2006).

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In addition, the mycotoxin DON stimulates the immune system when provided in

a chronic manner, as proven by increased leukocyte counts, and alters the or-

ganism’s acute immune response to LPS-induced endotoxaemia, mirrored in

modifications of clinical symptoms (Tesch et al. 2015). However, continuous

measurement of WBC counts is not always routine, thus chronic DON-exposure

often remains undetected although this provokes economic losses (e.g. reduced

weight gain) in pigs. DON, mainly produced by the fungi Fusarium graminearum

and F. culmorum, is often detected in wheat and maize. Both types of grain are

major components of porcine diets that frequently expose pigs, as the most

DON-sensitive species (Spate et al. 2004, Roth et al. 2014), to toxicologically

relevant DON levels. For that reason it can be assumed that a large proportion

of commercially used swine may suffer from chronic dietary DON exposure dur-

ing their lifetime, which can be co-exposed to LPS-releasing Enterobacteriaceae

species such as Salmonella, Campylobacter, and Escherichia coli (E.coli) de-

pending on modern husbandry conditions (Smith et al. 1968, Berends et al.

1996).

In veterinary practice the clinical examination and especially the rectal tempera-

ture measurement of pigs can be distorted by a large range of intrinsic stress

factors caused by the activation of the immune response on the one hand and

the unfamiliar handling with humans during clinical examination on the other

hand. Although rectal temperature is assumed to represent actual body core

temperature, it has to be emphasised that the rectum per se does not belong to

the body core. Therefore, in case of pathophysiological conditions, changes in

body core homeostasis may not be reflected entirely in rectally measured tem-

perature and inflammatory blood parameters, thus evaluating porcine health sta-

tus incorrect. In order to investigate the nature of the relationship between body

temperatures under physiological (clinically inconspicuous) as well as under

combined acute and chronic pathophysiological conditions, we used an experi-

mental model described earlier (Tesch et al. 2015), contrasting pigs chronically

pre-exposed with DON to control-fed pigs under clinically inconspicuous and

acute endotoxaemic conditions. The latter was induced either via the jugular or

portal vein and those different LPS-entry sites attempted to create two endotox-

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aemic conditions in both feeding groups based on their liver passage. Pre-

hepatic or portal infusion passes the liver first, while jugular-infused LPS spreads

through the systemic blood circulation before passing the liver. This crucial dif-

ference between both entry sites highlights the role of the liver as a major me-

tabolizing organ, also for xenobiotics such as LPS. Previously published data

have already shown an impact of DON-feeding and LPS-infusion on clinical

signs, fever response in body core temperature, leukocyte counts and TNF-α

(Tesch et al. 2015). In addition to these published results we complemented our

data with measurement of rectal temperature, Trp and Kyn analyses. Thus the

present study aims at determining relevant relationships between body tempera-

ture, their measurement methods and various inflammatory indicators in porcine

blood in clinically inconspicuous as well as in pathophysiological conditions, in-

duced by DON and LPS.

2. Materials and Methods

Experiment and procedures were conducted according to the European Com-

munity regulations concerning the protection of experimental animals and the

guidelines of the German Animal Welfare Act and were approved by the ethical

committee of the Lower Saxony State Office for Consumer Protection and Food

Safety (file number 33.4-42502-04-13/1274).

2.1. Experiment

The general experimental setup, including the infusion-protocol (experimental

groups), sampling and temperature measurement is depicted in Figure 1.

The present study was conducted using a total of 44 male castrated pigs (Ger-

man Landrace, Mariensee, Germany) with an initial mean body weight (BW) of

25.8 ± 3.7 kg. Animals were divided in two feeding groups (CON-diet and DON-

diet) and were fed restrictively twice a day over a four-week period with equal

quantities (2 × 700 g/pig and day; Figure 1). CON-diet contained 533 g/kg bar-

ley, 150 g/kg maize, 200 g/kg soybean meal, 50 g/kg rapeseed, 20 g/kg soybean

oil, 4 g/kg Lysine-HCl, 1.2 g/kg L-Threonine, 1.5 g/kg DL-Methionine, 10 g/kg

HCl-insoluble ash and 30 g/kg premix including minerals, trace elements and

vitamins, and the DON-diet comprised the same dietary components with the

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only difference that maize naturally contaminated with DON was exchanged for

non-contaminated maize. This resulted in a CON-diet containing 0.12 mg

DON/kg feed and a DON-diet containing 4.59 mg DON/kg feed (analysed by a

HPLC-method (Oldenburg et al. 2007).

On day 27 pigs were surgically equipped with arterial (Arteria carotis communis)

and venous catheters for infusions (Vena jugularis externa, V. splenica) and

sampling from post-hepatic area (V. jugularis interna) as well as from portal

drained viscera (V. portae hepatis; Figure 1). In addition, a temperature logger

for frequent body core measurement was incorporated to the abdominal cavity

and sutured on the right site of the intraabominal wall. Surgery took place under

sterile conditions and general anesthesia as described in detail previously

(Tesch et al. 2015). After a post-operative day of recovery (day 28) and the

morning feeding (06:45 - 07:00) on day 29 pigs were exposed to acute intrave-

nous treatments with physiological saline and 7.5 µg LPS/kg BW (E. coli

O111:B4, product number L2630, Sigma-Aldrich; LPS) for 60 min thereby creat-

ing six experimental groups in total.

Figure 1: Experimental design (upper panel, from (Bannert, Tesch et al. 2015, Tesch, Bannert et

al. 2015)) as well as the pattern of blood sampling and rectal temperature measurement (lower

panel).

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Control-infused groups (CON_CONjug.-CONpor., DON_CONjug.-CONpor.) received

physiological saline into jugular and portal vein, while LPS groups were portal

(CON_CONjug.-LPSpor., DON_CONjug.-LPSpor.) or jugular (CON_LPSjug.-CONpor.,

DON_LPSjug.-CONpor.; Figure 1) infused with LPS. Over a period of 210 min,

starting 30 min before infusion until 180 min post infusionem (p.i.) rectal and in-

traabdominal temperature were measured. Furthermore, serial blood samples

(Fig. 1) were taken for leukocyte counts (EDTA Monovette®, Sarstedt AG & Co.,

Sarstedt, Germany), TNF-α (Li-Heparin Monovette®, Sarstedt AG & Co., Ger-

many) and Kyn-Trp analyses (Serum Monovette®, Sarstedt AG & Co., Ger-

many). Collected blood samples for TNF-α, Trp and Kyn analyses were imme-

diately centrifuged at 2123 g for 15 min at 15°C (Varifuge 3.0 R, Heraeus Hold-

ing GmbH, Hanau, Germany) and aliquots of plasma and serum were stored at

-20°C until analysis.

In order to investigate the relationships between rectal and core body tempera-

ture and inflammatory markers under physiological and pathophysiological con-

ditions we defined our experimental groups in the following as “clinically incon-

spicuous conditions”, summarising the CON- and DON-fed, control-infused

groups (CON_CONjug.-CONpor., DON_CONjug.-CONpor.), and as “pathophysiologi-

cal conditions”, summarising the CON- and DON-fed, LPS-infused groups

(CON_CONjug.-LPSpor., CON_LPSjug.-CONpor., DON_CONjug.-LPSpor., DON_

LPSjug.-CONpor.).

2.2. Measurements and analyses

2.2.1. Body temperature

Body temperature was recorded intraabdominally (body core) and rectally using

two different measurement methods. Rectal temperature was measured at 13

points in time (-30, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 and 180 min)

with a digital thermometer (vet digital thermometer, Henry Schein Inc., Melville,

NY, USA) on the rectal mucosa. The thermometer was capable of measuring in

a total range of 32.0 - 42.9°C with an accuracy of ± 0.1°C within the limited

scope of 35.5 - 42.0°C. Intraabdominal temperature (Tesch et al. 2015) was

measured every 5 minutes (42 points in time) with a temperature logger (Ther-

mochron i-Button DS1921H-F5, Maxim integrated™, San Jose, CA, USA), fixed

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in the abdominal cavity. The logger was capable of measuring in a range of 15 -

46°C with an accuracy of ± 1.0°C and a resolution of 0.125°C.

2.2.2. Leukocyte counts

Serial whole blood samples, taken at 10 times (-30, 15, 30, 45, 60, 75, 90, 120,

150, 180 min), were analysed immediately after sampling by an automatic hae-

matology analyser (Celltac MEK 6400, Nihon Kohden Europe GmbH, Rosbach,

Germany) for total leukocyte counts. Additionally, two blood smears were pre-

pared of each blood sample and stained according to PAPPENHEIM in order to

differentiate the populations of leukocytes using oil immersion brightfield micros-

copy (Nikon Eclipse E200, Nikon GmbH, Tokyo, Japan) at a magnification of

1000 for differentiation of 100 leukocytes per slide (Tesch et al. 2015).

2.2.3. TNF-alpha

TNF-α concentrations in plasma were determined at six selected times (−30, 30,

60, 90, 120, 180 min) using a quantitative ELISA kit (Quantikine ELISA Porcine

TNF-α Immunoassay, level of detection 2.8 - 5 pg/mL, R & D System Inc., Min-

neapolis, MN, USA) and photometric sample measurement (Tecan® infinite

M200, Tecan Trading AG, Männedorf, Switzerland) at detection wavelengths of

450, 540, and 570 nm (Tesch et al. 2015).

2.2.4. Tryptophan, kynurenine

Trp and Kyn concentrations in blood serum were determined by HPLC after fat

extraction with n-hexane and protein precipitation using cold ethanol. After cen-

trifugation at 20800 g, the supernatant was quantitatively transferred into a flask

and evaporated in a nitrogen stream at 40°C. The residue was dissolved in

aqueous mobile phase A. After filtration (amcro filter, PVDF, 0.45 µm) 20 µL of

the filtrate were injected into a Shimadzu HPLC system (Shimadzu, Kyoto, Ja-

pan), consisting of two solvent pumps (LC-20AT), vacuum degasser (DGU-

20A3), autosampler (SIL-20AC HT), column oven (CTO-20AC), diode array de-

tector (SPD-M20A) and communication bus module (CBM-20A). Samples were

run through a C18 column (Inertsil ODS-2, 150 mm x 3 mm i.d., 5 µ) at a flow

rate of 0.5 mL/min and were eluted using a binary gradient system. Mobile

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phase A consisted of sodium 1-hexanesulfonate monohydrate (IPCC6; 10 mM)

and 0.5% (v/v) acetonitrile in ultrapure water, adjusted to pH 2.3 with 2 M phos-

phoric acid. Mobile phase B consisted of 100% acetonitrile. The detection wave-

lengths were 278 nm for Trp and 360 nm for Kyn.

2.2.5. Statistics

All statistics were performed by using the software packages of SAS Enterprise

Guide 6.1 (SAS Institute Inc., Cary, NC, USA, 2013) and Statistica 13 (StatSoft

Inc., 2015).

Data on rectal temperature were analysed using the SAS procedure “MIXED”

with an autoregressive covariance structure and group (six experimental

groups), time and their interactions as fixed factors. Effects were regarded to be

significant (adjusted Tukey post-hoc test) at a likelihood lower than or equal to

0.05, while tendencies or trends were discussed for probabilities higher than

0.05 but lower than 0.1.

Data on Trp, Kyn and Kyn-Trp ratio were analysed using the SAS procedure

“MIXED” with a compound symmetry covariance structure and group (six exper-

imental groups), sampling site (two locations: V. jugularis interna, V. portae

hepatis), time and their interactions as fixed factors. Effects were regarded to be

significant (adjusted Tukey post-hoc test) at a likelihood lower than or equal to

0.05, while tendencies or trends were discussed for probabilities higher than

0.05 but lower than 0.1.

The relationship between rectal and body core temperature as well as between

body temperatures (rectal and body core temperature) and leukocyte counts was

evaluated by linear regression for each experimental group. Additionally, the re-

lationship between temperature method (body core, rectal) and various inflam-

mation markers (leukocyte counts, TNF-α, Trp, Kyn, Kyn-Trp ratio) at selected

points in time was described with a Pearson correlation as was the relationship

in between the inflammatory markers. In order to analyse the respective rela-

tionship between the parameters, their common time points were included in the

statistical analysis, adjusted to each relationship.

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3. Results

3.1. Body core vs. rectal temperature

Under clinically inconspicuous conditions body core and rectal temperature re-

mained stable during the entire observation period, without any impact of DON.

However, rectal temperature (CON-diet: 38.8°C; DON-diet: 38.7°C) was on av-

erage 1.0°C lower than intraabdominal measurement (CON- and DON-diet:

39.7°C).

In accordance with the published intraabdominal data (Tesch et al. 2015), rectal

temperature measurement showed also a significant hyperthermia in LPS-

infused animals compared to control-infusion with an average increase of 1.5°C,

starting from 30 min p.i., irrespective of infusion site or diet. However, the

marked dietary effect found in body core measurement could not be confirmed

with the rectal temperature method.

Both methods, the intraabdominal and the rectal measurement, were significant

positively related (Tab. 1, Fig. 2) and the slopes of their linear regressions gen-

erally revealed that the increase in body core temperature per 1°C increase in

rectal temperature was approximately 25% higher in LPS-infused pigs (patho-

physiological conditions, 0.83°C abdominal per 1°C rectal increase on average)

compared to NaCl-infused pigs (clinical inconspicuous conditions, 0.66°C ab-

dominal per 1°C rectal increase), albeit the different treatments within the LPS-

infused pigs seemed to modify the magnitude of this difference (Tab. 1).

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Figure 2: Regression analyses for the relationship between individual mean values of rectal and

body core temperatures of CON- and DON-fed, control-infused (a) and LPS-infused (b) groups

over a period of 180 min (13 points in time). In clinically inconspicuous pigs (a; n = 13) the slope

of the linear regression line is almost equal, while pigs under pathophysiological conditions (b;

n = 27) showed significant differences (p = 0.02) between CON_CONjug.-LPSpor. and

CON_LPSjug.-CONpor. (Tab. 1).

Table 1: Linear regression of rectal temperature (x) on body core temperature (y) (y = m x + n)

over a period of 180 min (n = 40; 13 points in time).

Coefficient of determination (R²) is regarded to be significant * p < 0.05; slopes (m) with no com-

mon superscripts (a,b) are significantly different within line (p < 0.05).

3.2. Tryptophan, kynurenine

The concentration of Trp was analysed in serum samples from venous (portal

and jugular) blood at -30 and 180 min p.i.. In control-infused groups Trp levels

remained stable, while LPS induced a significant decrease in Trp concentration

from -30 to 180 min p.i. in group CON_CONjug.-LPSpor. and DON_LPSjug.-CONpor.

(V. jugularis p ≤ 0.01; V. portae p ≤ 0.1). Statistical main effects showed a signif-

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icant interaction between group, sampling site and time (p group × sampling site × time <

0.001).

Furthermore, the concentration of Kyn was analysed in the same blood samples

of venous (portal and jugular) blood at the same points in time (-30, 180 min

p.i.). In control-infused groups no differences were recorded, while LPS-infusion

caused a significant increase in Kyn values (p ≤ 0.001; CON_CONjug.-LPSpor. p =

0.07) from -30 (CON_LPSjug.-CONpor. 0.13 g/mL, DON_LPSjug.-CONpor. 0.15

µg/mL, DON_CONjug.-LPSpor. 0.1 µg/mL) to 180 min p.i. (CON_LPSjug.-CONpor.

0.31 µg/mL, DON_LPSjug.-CONpor. 0.27 µg/mL DON_CONjug.-LPSpor. 0.25

µg/mL). Considering the 180 min p.i. Kyn levels, group CON_LPSjug.-CONpor. had

significantly higher Kyn serum concentrations than both control-infused groups.

Statistical main effects showed a significant interaction between group, sampling

site and time (p group × sampling site × time < 0.001) as well as significant single effects

(p group = 0.006, p sampling site < 0.001, p time < 0.001).

Figure 3: Kyn-Trp ratio at -30 and 180 min p.i. from jugular and portal sampling site in all exper-

imental groups. LPS-infusion caused an increase in Kyn-Trp ratio (p < 0.001) from -30 to 180 min

p.i. while control-infused groups remained at their baseline level. Data represent LSmeans

(PSEM ± 0.004) and statistical main effects were distributed as followed: p group < 0.001, p sampling

site < 0.001, p time < 0.001, p group × sampling site × time < 0.001. Columns with no common superscripts

are significantly different within sampling site (portal: A, B or jugular: a, b) (p < 0.05).

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Kyn-Trp ratio as an important inflammatory maker was calculated from Trp and

Kyn measurements at -30 and 180 min p.i. for both sampling sites. The calculat-

ed ratio remained constant at a baseline level of 0.01 in control-infused groups,

while the LPS-infusion caused a significant increase in both sampling sites at

180 min p.i. (Fig. 3). There was no particular impact of dietary treatment on the

development of Kyn-Trp ratio.

3.3. Body temperatures vs. inflammation markers

Pearson correlations were evaluated between the temperature methods, Trp,

Kyn and Kyn-Trp ratio as well as leukocyte counts and TNF-α at five selected

points in time (15, 30, 60, 120, 180 min p.i.) due to the different time kinetics of

these parameters. Presentation of variables has been reduced to relevant corre-

lations which will be described and depicted in the following (Tab. 2).

With respect to relationships between body temperatures and inflammation

markers significantly negative correlations were found between temperature at

60 min p.i. as well as 180 min p.i. and leukocyte counts at 60 min p.i. (both sam-

pling sites). The correlation between body core temperature and their respective

leukocyte counts was less pronounced compared to those with rectal tempera-

ture, reflected in their lower r-values.

A strong positive correlation was detected between body temperature and TNF-α

concentrations, most pronounced at 60 min p.i. and with a bit lower r-values at

180 min. Here also a stronger correlation between TNF-α and rectal temperature

was reflected in the higher r-value compared to body core temperature.

Moreover, relationships between temperatures (body core and rectal) at 60 min

p.i. as well as 180 min p.i. and Kyn at 180 min p.i. (both sampling sites) were

positively correlated. In addition to Kyn, significantly positive correlations were

also found between Kyn-Trp ratio at 180 min p.i. (both sampling sites) and body

temperatures. However, body core measurement at 60 as well as 180 min p.i.

showed again a greater scattering (e.g. r = 0.492 for body core temperature and

Kyn-Trp ratio (V.portae) at 180 min p.i.) compared to the corresponding relation-

ship between Kyn-Trp ratio and rectal temperature (r = 0.643).

Considering relationships amongst the inflammation markers itself, significantly

negative correlations were determined between leukocyte counts at 60 min p.i.

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Tab

le 2

: P

ears

on

corr

ela

tio

n m

atr

ix f

or

sele

cte

d p

ara

mete

rs inclu

din

g a

ll exp

erim

enta

l gro

ups a

t diffe

rent po

ints

in t

ime.

r-valu

es in

red a

re s

ignific

ant (p

≤ 0

.01).

Paper II

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and TNF-α at 60 and 180 min p.i. as well as Kyn-Trp ratio at 180 min p.i. (both

sampling sites). Furthermore, at 60 and 180 min p.i. TNF-α (both sampling sites)

was positively well correlated with Kyn-Trp ratio at 180 min p.i. (both sampling

sites; Fig. 4).

Looking at the dot-plots into more detail (Tab. 2), signifying the relationships be-

tween the respective parameters, it became clear that the data points were

strongly clustered into two groups, NaCl-infused and LPS-infused animals, which

biased correlation analysis.

Figure 4: Correlation between TNF-α and Kyn-Trp Ratio at 180 min p.i. (portal sampling site).

Experimental groups are marked in different colours, with NaCl-infused groups near baseline and

LPS-infused groups showing increased TNF-α and Kyn-Trp ratio values.

Thus, regression analysis was performed for each experimental group, distin-

guishing between healthy and endotoxaemic pigs (Fig.5, Table 3). The regres-

sion analyses between rectal temperature and leukocyte counts from 15 to 180

min p.i. under clinically inconspicuous conditions showed high individual scatter-

ing and no significant relationships (Fig. 5a). In contrast, regression analysis be-

tween body core temperature and leukocyte counts revealed a significant rela-

tionship in DON-fed group (Tab. 3).

Under pathophysiological conditions significant relationships between both tem-

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perature measurement methods and leukocyte counts from 15 to 180 min p.i.

were found in all LPS-infused animals. Due to differences in the slope, the linear

regression line (Fig. 5b) was significantly steeper (p ≤ 0.01) in CON_LPSjug.-

CONpor. (4.12 x 10³/µL decrease in leukocyte counts per 1.0°C increase in rectal

temperature) compared to CON_CONjug.-LPSpor. (2.01 x 10³/µL decrease in leu-

kocyte counts per 1.0°C increase in rectal temperature), irrespective of blood

sampling site and temperature measurement (Table 3). In addition, comparing

the relationship between leukocyte counts and body core temperature, group

CON_LPSjug.-CONpor. showed also a significantly steeper (p < 0.01) slope than

DON_CONjug.-LPSpor. (Tab. 3).

Table 3: Linear regression analyses of either rectal or body core temperature (x) on leukocyte

counts in V. portae hepatis (y) (y = m x + n) over a period of 15 - 180 min p.i. (n = 39; 9 points in

time).

Data represent coefficient of determination (R²) and were regarded to be significant at * p ≤ 0.05;

m-values with no common superscripts (rectal temperature: A,B,C; body core temperature: a,b,c)

are significantly different within line ( p < 0.05).

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Figure 5: Regression analyses for the relationship between the mean values of rectal tempera-

ture and leukocyte counts (portal sampling site) of CON- and DON-fed, control-infused (a) and

LPS-infused (b) groups over a period of 15 - 180 min (9 points in time). In clinically inconspicu-

ous pigs (a; n = 13) the regression showed no significant relationship. Under pathophysiological

conditions (b; n = 26) significant differences (p ≤ 0.01) in the slope of the linear regression line

between CON_CONjug.-LPSpor. and CON_LPSjug.-CONpor. was found.

4. Discussion

Available literature on relationships between different body temperature meas-

urement sites primarily reports on single effects of selected abiotic and biotic

environmental conditions on the mammalian body temperature neglecting intrin-

sic temperature variations in dependence on mammals´ state of health such as

ambient temperatures, physical exercise and disease. The same applies to liter-

ature on relationships between body temperature and inflammatory markers in-

cluding variations in mammalian state of health. For that reason, we conducted

an experiment to answer the following questions: firstly, does rectal temperature

constantly reflect anatomical body core temperature in pigs under physiological

and different pathophysiological conditions (acute local (portal) or systemic

(jugular) LPS and chronic DON inflammation)? Secondly, how does body tem-

perature correlate to selected inflammatory markers, caused by a broad spec-

trum of triggering conditions?

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Under clinically inconspicuous conditions body temperatures as well as the ex-

amined inflammation markers remained within physiological levels indicating the

absence of an acute inflammatory response in 0.9 % NaCl treated pigs. In CON

and DON-fed pigs both temperature measurement sites revealed a constant re-

lationship characterised by an average temperature difference of about 1.0°C

and a temperature increase of 0.64 - 0.68°C intraabdominal per 1.0°C rectal.

These results lead to the assumption that rectally measured temperature cannot

be equated with those measured in the body core and that the temperature ho-

meostasis in the abdominal cavity is regulated more strictly than that in the pe-

ripheral, rectal localisation. But, depending on their relatively constant relation-

ship, the deduction of the abdominal core temperature from the rectally meas-

ured one seems to be a valid method in clinically inconspicuous pigs.

Confirming our results under physiological conditions, similar close relationships

were described in mice and rabbits between temperatures measured via subcu-

taneous transponder and rectally used digital thermometer (Hartinger et al.

2003). In addition, comparing measurements of femoral arterial (caudal abdo-

men) and rectal temperature showed a mean difference of 0.7°C in pigs (Han-

neman et al. 2004). This slightly lower temperature difference might result from

the actual anatomical localisation of the femoral arterial catheter being not com-

pletely in the centre of the abdominal cavity but in the caudal region. In contrast

to that, studies on humans equated body core and rectal temperature due to

considerably lower temperature differences of ≤ 0.3°C between the pulmonary

artery (PA) and the rectum on average (Fulbrook 1993, Henker et al. 1995,

Greenes et al. 2004). Referring to the temperature differences between femoral

artery and the rectum (Hanneman et al. 2004), the comparatively small variation

between PA and rectum could be explained by the anatomical localisation like-

wise. The PA is located in the thorax, which is generally regarded as part of the

body core, but still differs from the anatomical centre of the body in the ab-

dominal cavity in two major points. Firstly, the PA is closely located to the lungs,

where the temperature of the inhaled air always needs to be equilibrated and

one could assume that there is always a slightly lower temperature and secondly

the metabolic heat production in liver, small and large intestine in the abdomen

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ensures a constant high temperature (Durotoye et al. 1971) in contrast to the

lower metabolic activity in the thoracic cavity. The results of an experiment on

generally anaesthetised pigs indicated only small differences of about 0.4°C be-

tween hepatically and rectally measured temperatures (Dickson et al. 1979),

seemingly contradicting the aforementioned relationship between temperature

and metabolically active abdominal organs. However, this observation was made

under general anaesthesia, whereas our data were evaluated in conscious pigs.

Because sedatives and anaesthetics such as isoflurane affect the centrally con-

trolled physiological thermoregulation negatively (Roth et al. 2014, Tansey et al.

2015) by peripheral vasodilatation (Imrie et al. 1990, Buggy et al. 2000), the low-

er temperature difference between liver and rectum reported in this study (Dick-

son et al. 1979) can be thus explained.

With respect to the impact of DON-feeding on the explanatory power of regres-

sion analyses, there is currently no literature reporting comparable results. How-

ever, DON is known to modify the immune system and thus could influence body

temperature. While the present study as well as previously published research

did not show an effect of sole DON-feeding on thermoregulation in apparently

healthy swine (Dänicke et al. 2013, Tesch et al. 2015), an oral DON-exposure of

0.75 - 3 mg DON/kg diet for 28 d induced a slight decrease in skin temperature

within seven days (Rotter et al. 1994). Similar results were published for wild

type mice, showing a decline in body core temperature up to 72 h after oral ad-

ministration of a single DON dosage of 6.25 - 25 mg/kg BW (Girardet et al.

2011). Although the present study showed neither clinical signs nor alterations in

body temperature in DON exposed pigs, increased leukocyte counts in DON-fed

pigs indicated the activation of the immune system (Tesch et al. 2015). The re-

gression between leukocyte counts and body temperature reflected this change

to some extent: leukocyte counts were significantly related to body core temper-

ature in DON-fed pigs, albeit with a rather low R2-value. In contrast, in CON-fed

pigs this relationship had an even lower R2-value and was not significant.

Under pathophysiological conditions we found typical signs of a systemic in-

flammatory response characterised by marked fever accompanied by severe

leukopenia, and an increase in pro-inflammatory major cytokine TNF-α. Addi-

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tionally, the increase in Kyn-Trp ratio resulting from the LPS induced IDO activi-

ty, which enhanced Trp catabolism to its intermediate Kyn (Moffett et al. 2003,

Schrocksnadel et al. 2006) is also an indicator for immunological activity. All

LPS-infused groups showed a significant correlation of both temperature meas-

urement methods, but with varying explanatory power, indicated by the different

slope of the linear regression line, compared to clinically inconspicuous pigs. In

CON-fed groups the slope of portal-infused pigs (m = 0.97) was significantly

greater as those of jugular-infused (m = 0.69) counterparts. Although this ten-

dency was also visible in DON-fed groups (portal vs. jugular: 0.87 vs. 0.77), we

could not detect any statistical significance. This might be explained by an al-

tered reaction of the organism to the various inflammatory situations and con-

firms a dependence of the immune response to the point of endotoxin entry as

well as mycotoxin pre-exposure (Tesch et al. 2015). Portal entry of LPS resulted

apparently in an enlargement of the actual temperature body core towards the

rectum, as a change of 1°C rectally also showed a near-equal change in-

traabdominally (0.97°C, 0.87°C). In jugular-infused pigs the temperature body

core appeared to be maintained as the rectal change of 1°C yielded only a

change of 0.69°C (CON-fed) and 0.77°C (DON-fed), respectively. We suggest

that LPS-infusion via portal route resulted in a larger hepatic activity due to met-

abolic stress (Brock et al. 1975, Dickson et al. 1979) compared to jugular infu-

sion, resulting in an enlargement of the body core temperature region.

In addition, porcine as well as human febrile response has been characterised

by slow and blunted changes in rectal temperature compared to skin tempera-

ture (Siewert et al. 2014) or arterially measured body core temperature (Greenes

et al. 2004). While the difference between rectal and PA measurement is stated

with rectal temperature 0.1 - 0.2°C above PA in human intensive care patients

(Fulbrook 1993, Rotello et al. 1996), data from porcine intensive care patients

showed a difference between rectal and femoral artery with arterial values being

0.2 - 0.4°C above rectal ones (Hanneman et al. 2004). These findings sustain

that body core temperature homeostasis cannot be maintained in certain condi-

tions. In intensive care this is presumably based on the variety of influencing fac-

tors like mechanical ventilation, respiratory infections or antibiotics, while our

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results are most likely due to a local effect. The more rapid increase in liver tem-

perature compared to changes in temperature at other body sites under patho-

physiological conditions has also been shown before in pigs (Dickson et al.

1979) and humans (Brock et al. 1975). The lower febrile response in DON-

exposed pigs compared to their CON-fed counterparts (Tesch et al. 2015) leads

to the assumption that DON modulated thermoregulatory mechanisms by atten-

uating the action of LPS, indicating a greater capacity to successfully counteract

LPS after DON pre-exposition.

Additionally, looking at the results of regression analyses between rectal tem-

perature and leukocyte counts, we also found the mentioned differences based

on LPS infusion-site in CON-fed groups. The decrease in leukocyte counts per

1°C increase in body temperature is significantly stronger in jugular-infused

compared to portal-infused pigs. This is probably explained by the fact that a

portal-infused immune stimulus (LPS) entirely has to pass the liver first (first-

pass effect) before reaching systemic blood circulation. Thus, the liver could

proportionally remove the endotoxin and reduce the pressure for the rest of the

organism, especially for the circulating leukocytes, while a jugular-infused path-

ogenic agent spreads through the whole blood system before passing the liver.

This assumption is also supported in clinical signs of CON-fed pigs, where por-

tally LPS-infused animals showed an earlier return to physiological levels com-

pared to jugular-infused pigs (Tesch et al. 2015). Moreover, in our pigs rectal

temperature seems to be more suitable for correlating with inflammatory markers

than body core measurement. This difference in the strength of the relationships

might result from the smaller standard deviation of the rectal measured tempera-

ture (SD ± 0.65) compared to that of the body core measurement (SD ± 0.73).

The higher standard deviation of the latter method could be a consequence of

the rapid and much greater heat production in the body core in portal-infused

experimental groups (Brock et al. 1975, Dickson et al. 1979) and thus a reflec-

tion of a faster and more sensitive reaction to a stimulus in the body core com-

pared to the rectum. Other studies reported similar results in pigs under general

anaesthesia with rectal (SD ± 0.7), oesophageal (SD ± 1.1) and pharyngeal (SD

± 1.0) measurement (Musk et al. 2016).

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Considering the entire range of observed clinical inconspicuous and conspicu-

ous conditions, we found a close correlation between alterations in body tem-

perature, as one of the most important symptoms for clinical monitoring, and in-

flammatory markers (leukocyte counts, Kyn, Kyn-Trp ratio, TNF-α). Regarding

the respective r-values, leukocyte counts and TNF α, as one of the major fever

inducing cytokines (Mackowiak 1998, Roth et al. 2014), showed the closest rela-

tionship with body temperature, followed by Kyn or Kyn-Trp ratio to a lesser ex-

tent. Although the latter relationships are less strong, Kyn and especially Kyn-

Trp ratio are reported to have a prognostic potential in the prediction of post-

traumatic sepsis in human patients (Logters et al. 2009). Thus, this potential

probably also applies to endotoxaemic conditions, such as in our LPS-infused

pigs. The detected relationships between Kyn-Trp ratio and other inflammatory

markers (leukocyte counts, TNF-α) as well as body temperature supports the

assumption that this parameter is an important diagnostic interlink for an early

detection of the immune response in septic and endotoxaemic patients in veteri-

nary medicine as well.

This is further substantiated by the strong connection between inflammation

markers, confirming a close association. The closest relationship was detected

for TNF-α and leukocyte counts with high r values (> 0.82), closely followed by

TNF-α and Kyn-Trp ratio, irrespective of blood sampling site (jugular or portal).

This reveals that leukocyte counts are pivotal in endotoxaemic specimens, being

strongly related to both body temperature and TNF α, a major pyrogen.

Clinical research in humans investigated the interactions between body tempera-

ture and inflammatory markers confirms our results, describing the relationship

between rise in rectal temperature and TNF-α (Rhind et al. 2004,) as well as leu-

kocyte counts (Cooper et al. 2010). The latter study supports our assumption

that total leukocyte counts seemed to be highly sensitive for changes in mamma-

lian organisms, finding an increase in WBC counts under warm and cold envi-

ronmental conditions whereas TNF-α did not change in cool trial but increased

only in warm environment. Moreover, correlation between rectal temperature

and leukocyte counts was more pronounced than that to TNF-α.

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In conclusion, the present study showed that the relationship between in-

traabdominal and rectal temperature is not constant under all (patho)physio-

logical conditions (Robinson 2004) and thus extrapolating from rectal directly to

body core temperature requires caution. Particularly the different endotoxaemic

situations yielded highly variable relationships between both temperature sites,

hinting at a change in expansion of core temperature in the body. Furthermore,

body temperature was strongly correlated to leukocyte counts, which in turn

showed a strong relationship to TNF-α, indicating the pivotal role for leukocyte in

endotoxaemia. Therefore, leukocyte counts appear to be a good prognostic tool

in practice besides the usual use as indicator for an infection, providing insight

into TNF-α development without the necessity for actual measurement of the

latter, which is costly and time-consuming. Such an extended diagnostic could

also help to identify clinically inconspicuous animals and thus aid in reducing

economic losses caused by clinically inapparent pigs.

Acknowledgments: The authors would like to thank Nicola Mickenautsch, Elenia

Scholz, and Lara Lindner of the Institute of Animal Nutrition, Friedrich-Loeffler-Institute

in Braunschweig, Germany, for their excellent technical support in sample analysis and

the “Deutsche Forschungsgemeinschaft” (DFG) for financial support (DA 558/1-4).

Moreover, special thanks to Marc-Alexander Lieboldt for his help in editing the manu-

script.

Author Contributions: The project was conceived and designed by Jeannette Kluess,

Jana Frahm, Susanne Kersten, Gerhard Breves, Hermann-Josef Rothkötter and Sven

Dänicke while Tanja Tesch, Erik Bannert, Jeannette Kluess, Jana Frahm, Lydia Renner,

Stefan Kahlert and Sven Dänicke organized and performed the experiments including

the sample collection. Tanja Tesch, Jeannette Kluess, Jana Frahm, Liane Hüther and

Sven Dänicke analysed and interpretated the data. The manuscript was written by Tanja

Tesch whereas Erik Bannert, Jeannette Kluess, Jana Frahm, Liane Hüther, Susanne

Kersten, Gerhard Breves, Lydia Renner, Stefan Kahlert, Hermann-Josef Rothkötter and

Sven Dänicke were revising the paper.

Conflicts of Interest: The authors declare no conflict of interest.

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Paper II

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General discussion

- 76 -

General discussion

In swine production a contemporaneous exposure of the porcine organism with

DON and LPS is not unusual due to the widespread mycotoxin-contamination of

cereal grain in northern temperate climate and the high infectious pressure in

farm animal production by gram-negative bacteria (Padoan 2016). In this context

the detection of intoxications as early as possible is of crucial interest for keeping

the stock healthy and thus minimise economic losses in swine population. Nev-

ertheless, research about the interaction between DON and LPS, especially with

regard to the role of the liver in toxin elimination as well as possible modulating

effects on the APR and specific immune parameters in pigs are scarce.

For that reason, we conducted a study elucidating possible liver-related modula-

tory effects on APR in pigs that are co-exposed to chronic oral DON and acute

venous LPS-intoxication. Thereby we specifically focussed on the kinetics and

manifestations of clinical symptoms and different immune-related blood parame-

ters, depending on hepatic (portal) or systemic (jugular) LPS-infusion in combina-

tion with diet (Paper I & II).

Figure 6: Experimental timeline showing four weeks of DON-feeding followed by an hour of LPS-

exposure as well as constant sampling and clinical observation up to 180 min p.i. on day 29.

Furthermore, the different time ranges of responses to DON, LPS or rather their interactions are

marked.

General discussion

- 77 -

Regarding to our experimental design, toxin impacts are divided according to

their chronological occurrence in (Fig. 6): response of porcine organism to sole

DON-feeding (until day 29), peracute (from 0 to 60 min p.i.) and acute (from 60 to

180 min p.i.) response to LPS-infusion as well as toxin-interactions and the he-

patic impact (from 0 to 180 min p.i.).

For completing published kinetics (Paper I) and selected correlation and regres-

sion analyses (Paper II) a PCA with additional data (GLU, TB, LAC, pO2, pCO2,

BE, (Bannert, Tesch et al. 2015); ASAT (Renner, Kahlert et al. 2017), LPS (un-

published data)) was done to emphasise the explanatory power as well as varia-

tion and potential relationships between different clinical and blood parameters.

The illustrated biplot (Fig. 7) with PC 1 and PC 2 depicts the explained variance

(correlation, R2) of mentioned parameters, each including the data of all six ex-

perimental groups. Whereby, the closer a parameter is located to 1.0/-1.0 the

better its variance can be explained by PC 1 and 2. Additionally associations be-

tween parameters, located near (e.g. body temperatures) or rather opposite (e.g.

leukocyte counts and rectal temperature) to each other, are reflected in this

analysis. In total, PCA explain 34.73% of the total variance of the original dataset

with the first two PCs. Corresponding to PCA the measurements of each pig,

subdivided in treatment group and sampling points in time, are illustrated in scat-

terplot (Fig. 8). This clearly shows the dependence of time while the effect of

treatment remains limited.

In practise, pigs chronically exposed to DON-contaminated diets show typical but

also unspecific and slow appearing symptoms such as reduced feed consump-

tion and live weight gain (Bergsjo, Matre et al. 1992, Bergsjo, Langseth et al.

1993, Dänicke, Valenta et al. 2004, Pestka and Smolinski 2005). Due to a re-

strictive feeding management, we were able to prevent this distinction in body

condition between DON- and CON-fed groups in our pigs. Even though no exter-

nal symptoms were observable, our results show the activation of the immune

system by an increase in leukocyte counts as the first detectable impact of sole

DON-feeding (Paper I). This DON-impact was also reported after 33d of DON-

exposure (Chaytor, See et al. 2011) as well as in in vivo data with a higher

General discussion

- 78 -

stimulation of lymphocytes after 9 weeks of DON-exposure (Malovrh and

Jakovac-Strajn 2010). Admittedly, other studies reported reverse DON-effects in

pigs and rodents, showing leukopenia after toxin exposure (Bondy and Pestka

2000, Kluess, Kahlert et al. 2015).

In contrast to the slow and clinically inconspicuous reaction of the porcine organ-

ism to DON, the first implications of LPS were immediately and clinically apparent

Figure 7: PCA explaining 34.73% of the total variance of the original dataset with the first two

PCs. The plotted variables contain the data from all experimental groups at each sampling point

in time (max. 13). Parameters located in the left quadrants showing increased values during the

experiment while the ones located in the upper right quadrant decreased. The closer they are

situated to circle line the better their variance is explained by PC 1 and 2. Variables are located

near or rather opposite to each other indicate close connections between these parameters.

General discussion

- 79 -

Figure 8: Scatterplot (corresponding to PCA, Fig. 7) illustrates the data of each pig, subdivided in

experimental group and sampling points in time (max. 13) by colour gradient from light to dark

according to groups labelling colour. Control-infused groups and data from early sampling points

in LPS-infused animals are mainly clustered in the right quadrants in the positive sector of Varia-

ble 1 and around zero line of Variable 2. In further time course LPS-infused pigs firstly cluster in

the upper left quadrant followed by an increasing individual diversification as well as poor group

differences, detectable towards the end of experiment (180 min) especially in the lower left quad-

rant.

15 min p.i. by for instance cyanosis, reddened eyes, tachycardia and tachypnea

(Paper I, (Dänicke, Brosig et al. 2013)).

Considering the peracute clinical symptoms we observed in all LPS-infused ani-

mals (Paper I; fever, cyanosis, reddened eyes, tremor, increased respiratory

General discussion

- 80 -

rate and difficulties), all variables are located in the upper left quadrant of PCA

(Fig. 7, dark blue and green coloured). They are negatively correlated with PC 1

and positively with PC 2, whereby the first PC (≥ - 0.5, except of tremor = - 0.4)

mainly explains the variance of the mentioned parameters. Thus, PCA confirms

the importance of selected clinical signs with a coherent time sequence in endo-

toxaemic pigs emphasised in Paper I.

However, increased respiration or rather respiratory difficulties did not show a

consistent time course (Paper I) and had a high individual variability (Fig. 10),

theirs arrangements in PCA indicate a great importance due to endotoxaemia

too. Whereas parameters, as nystagmus, dermographism, teeth gnashing, vom-

iting and retching, that were only sporadically observed or showed a marked in-

dividual variability within our experimental groups are probably of minor rele-

vance in porcine APR during LPS-intoxication. This hypothesis is supported by

PCA, where these parameters are located close to zero line in the upper and

lower left quadrant (PC 1 ≤ - 0.3, PC 2 ≤ 0.3) and thus most of the variance can-

not be explained by the first two PCs.

Nevertheless contrasting results, with dermographism observed in all LPS-

exposed pigs, were reported in other studies with a similar experimental setup

(Dänicke, Brosig et al. 2013).

In addition to the importance of single parameters, PCA also indicates close

connections between different variables. Considering for instance the relation-

ship between rectal temperature and clinical score (recap of relevant clinical

signs, Paper I), both triggered by cytokine release, their positioning in PCA con-

firms a positive correlation. The scatterplot also shows this close connection

(Fig. 9, rectal temperature - clinical score r = 0.59), whereby control-infused

groups cluster in physiological range of rectal temperature and lowest clinical

scores, while LPS-exposed animals display time-dependent increasing values in

both, temperature and clinical score. Nevertheless, this depiction indicates the

individual diversification within experimental groups resulting in enlarged vari-

ance.

General discussion

- 81 -

Figure 9: 3D-Scatterplot shows the close relationship between rectal temperature and clinical

score (r = 0.59) with the data of each pig from - 30 to 180 min p.i.. Control groups are clustered

in physiological levels of rectal temperature and lowest clinical scores, while LPS-groups show

increasing values during observation period. Further on individual scattering in CON-fed animals

appears to be higher compared to DON-fed ones.

In contrast to these parameters with coherent kinetics, the correlation between

respiratory rate and rectal temperature (Fig. 10, respiratory rate - rectal tempera-

ture r = 0.19) noticeably confirms the discontinuous kinetics of respiration as well

as the marked individual variations within experimental groups.

The occurrence of clinical symptoms mainly result from leukocyte-released in-

flammatory mediators as well as accompanied alterations, such as centralisation

General discussion

- 82 -

Figure 10: 3D-Scatterplot shows the data of respiratory rate and rectal temperature (r = 0.19) of

each pig from - 30 to 180 min p.i.. Control groups are mainly clustered in physiological levels of

rectal temperature while respiration shows noticeable individual variations. LPS-groups show

increasing values in both parameters during observation period as well as high individual scatter-

ing too.

of the blood circulation to vital organs (Schumer 1984), initially decreased cardi-

ac output (Hurtado, Gutierrez et al. 1992), altered respiratory rate and depth

(Paper I, (Hurtado, Gutierrez et al. 1992)) or increased tissue oxygen consump-

tion (Boekstegers, Weidenhofer et al. 1994). Along with this and the rapidly fol-

lowing counter regulation to restore homeostasis our results also show peracute

General discussion

- 83 -

LPS-impacts in related blood parameters (Paper I, (Bannert, Tesch et al. 2015)).

The first detectable alteration in these parameters we found is a significant de-

crease in WBC counts, starting 15 min p.i. (Paper I). In PCA leukocyte counts

(Fig. 7, dark red coloured) are located in the upper right quadrant showing a pos-

itive correlation with both, PC 1 and 2. In this case, as in clinical signs, the vari-

ance is mainly explained by the first PC (= 0.8). The opposite arrangement of

WBC counts and relevant clinical signs as well as body temperatures confirm

their close relationship due to well-known pathophysiological interactions during

APR. Furthermore, PCA also indicates the close connection between leukocyte

counts and TNF-α, which is located among variables of clinical signs in the upper

left quadrant (Fig. 7, pink coloured). TNF-α increases from 30 min p.i. (Paper I)

and is the first pro-inflammatory cytokine in APR activated cascade (Dauphinee

and Karsan 2006, Wyns, Plessers et al. 2015). Thus its arrangement is con-

sistent to the central role as pyrogenic (Fig. 7, positive correlation between TNF-

α and body temperatures) and pro-inflammatory mediator inducting endothelial

damage and permeability changes (Fig. 7, positive correlation between TNF-α

and hyperaemic conjunctivae/injected episcleral vessels) shown in other studies

(Morrison and Ryan 1987, Mackowiak 1998, Dänicke, Brosig et al. 2013, Roth

and Blatteis 2014, Wyns, Plessers et al. 2015).

The APR based alterations in metabolism and the related need of energy are

reflected, for instance, in glycolytic pathway. In general, the peracute enhanced

glycolysis in endotoxaemia lead to an initial hyperglycaemia followed by eu- and

hypoglycaemia (Bannert, Tesch et al. 2015) as already shown in other LPS stud-

ies (Lang, Spolarics et al. 1993, Maitra, Wojnar et al. 2000). In PCA GLU is locat-

ed near zero lines of PC 1 and 2 (Fig. 7, pink coloured), thus most of its variance

cannot be explained by both PC´s. This is probably due to its poor verifiability

based on the strict and rapidly regulation (Tirone, Brunicardi 2001). Furthermore,

our pigs showed a more pronounced LPS-effect in portal blood compared to jug-

ular samples. This result supports other reported findings, showing a hypoperfu-

sion of the GIT and decreased portal GLU uptake during endotoxaemia

(Whitworth, Cryer et al. 1989, Vallet, Lund et al. 1994, Amador, Garcia-Herrera

et al. 2007, Garcia-Herrera, Marca et al. 2008). However, the concurrently

General discussion

- 84 -

heightened oxygen consumption of other tissues is macroscopically and micro-

scopically shown by intrahepatic hyperperfusion in our pigs (Renner, Kahlert et

al. 2017) as well as in previous porcine and human studies (Gutierrez and Wulf

1996, Stanek, Reinhardt et al. 2012). Further on in our study, this is also proofed

by decreasing BE and pO2 levels from 30 min p.i. (Bannert, Tesch et al. 2015). In

PCA both variables BE and pO2 can be found in the upper right quadrant (Fig. 7,

light blue), with an almost equal positive correlation to PC 1 and 2. However, BE

seems to be a more relevant parameter (PC 1 = 0.6, PC 2 = 0.4) compared to

pO2 (PC 1 = 0.2, PC 2 = 0.25). This difference in explainable variance could pos-

sibly be explained by the more uniform kinetic of BE compared to pO2, that initial-

ly decreases in venous and arterial blood followed by a compensatory increase

in the arterial system only. Additionally, BE is a reflection of different variables

like pCO2 and pH that affect the acid-base balance and contains information re-

lating to the type of metabolic imbalance (Irizarry and Reiss 2009, Irizarry and

Reiss 2009). Regarding to this, PCA suggest a strong relationship between BE

and pH as they are located near to each other, while the possible relation to

pCO2 is substantially lower (Fig. 7, light blue). The pCO2 level showed no altera-

tions in venous system due to LPS but decreasing values in arterial blood from

120 min p.i. (Bannert, Tesch et al. 2015). This acute and thus slower response in

pCO2 compared to pO2 is consistent to its positioning near zero lines in PCA (Fig.

7, PC 1 and 2 = - 0.15). Nevertheless, PCA reflects the physiological relationship

between pCO2 and pO2.

In line with decreasing BE and pH values, the blood lactate level increases from

75 min p.i. onwards (Bannert, Tesch et al. 2015). In PCA this variable is located

in the lower left quadrant and negatively correlated with both, PC 1 and 2 (Fig. 7,

PC 1 = - 0.6, PC 2 = - 0.45). The opposite positioning of BE, pH and LAC confirms

their negative relationship. In previous studies it has been suggested that the

LPS-induced lactic acidosis (Bannert, Tesch et al. 2015) is caused by an ampli-

fied lactic output of leukocytes due to increased anaerobic glycolysis (Haji-

Michael, Ladriere et al. 1999). This is consistent with the severely affected leu-

kocytes and enhanced glycolysis due to LPS, observed in our study. Further, it

has been assumed that during APR the rate of pyruvate formation exceeds the

General discussion

- 85 -

oxidative capacity of mitochondria, inducing an accumulation of pyruvate, and

consequently, an increase in LAC formation (Robinson 1993). This is also poten-

tiated by a decrease in LAC utilisation (Levraut, Ciebiera et al. 1998).

Taking in to consideration pO2, pCO2, pH and LAC, we deduced that firstly acido-

sis was caused by decreased pCO2 (respiratory acidosis) followed by a metabolic

acidosis originated from increased LAC levels (McLellan 1991). Notwithstanding

Figure 11: 3D-Scatterplot shows the data of LAC and respiratory rate (r = 0.07) including each

pig from - 30 to 180 min p.i.. Control groups are clustered in physiological levels of LAC, while

values of respiratory rate are scattering. LPS-groups show increasing values during observation

period, with a cluster in physiological levels of LAC due to its kinetic.

General discussion

- 86 -

of the clear link between respiration and lactic acidosis, the scatterplot indicates

no significant relationship between these two parameters (Fig. 11, lactate - res-

piratory rate r = 0.07) in LPS-infused pigs. The depiction shows increasing values

of both but also great individual scattering. The lack of correlation is possibly due

to this great individual spreading as well as their temporal sequence. While LAC

increases towards the end of observation period, respiratory rate is markedly

unstable over the entire time.

Figure 12: Scatterplot shows the relationship between LAC and clinical score (r = 0.40) including

values of each pig from - 30 to 180 min p.i.. LAC values are mainly clustered in physiological

levels due to its late increase while clinical signs show a direct response to LPS-infusion seen in

increased score values during observation period.

General discussion

- 87 -

Irrespective of the latter connection, our data show a relationship between LAC

and clinical signs, whereby the scatterplot confirms the mentioned late increase

in LAC by clustering in physiological range with simultaneously spreading in clini-

cal score.

In human septic and infectious studies LAC has been established as prognostic

parameter for the severity or rather mortality (Trzeciak, Dellinger et al. 2007,

Rocha, Pessoa et al. 2013). Considering the scatterplot (Fig. 12) our results

show the highest individual values of LAC and clinical score in group

CON_LPSjug.-CONpor.. Whereas from a statistical point the highest levels of LAC

(Bannert, Tesch et al. 2015) and a significant earlier increase in clinical signs

were found in group DON_LPSjug.-CONpor. compared to CON_LPSjug.-CONpor. (Pa-

per I). But contrasting to LAC-prognosis pigs of DON_LPSjug.-CONpor. showed a

more rapid onset of symptom relief.

Apart from the fact that our pigs were slaughtered 195 min after beginning of

LPS-infusion and thus a prognosis about mortality or outcome of endotoxaemia

was impossible, our findings are generally in accordance with human research.

Similar to LAC, in human medicine KYN and KYN-TRP ratio are also used as

prognostic parameters in septic patients (Lögters, Laryea et al. 2008). They in-

crease due to the degradation of TRP to its metabolite KYN by IDO and TDO.

Thereby, the IDO activity that is characterised by the KYN to TRP ratio, that is

closely associated with the immune response. Corresponding results were found

in our LPS-infused pigs 180 min p.i. (Paper II). In PCA KYN and KYN-TRP ratio

are located nearby LAC and oppositely to TRP in the lower left quadrant (KYN PC

1 and 2 = - 0.4, KYN-TRP ratio PC 1 = - 0.5, PC 2 = - 0.6). This verifies the nega-

tive dependency of KYN and TRP. With relation to the prognostic potential of

KYN-TRP ratio our results in scatterplot (Fig. 13) show a marked relationship be-

tween KYN-TRP ratio and clinical score (r = 0.66) with group CON_LPSjug.-CONpor.

showing highest values, as in LAC and clinical score (Fig. 12). In reference to our

results and in agreement with published human data (Logters, Laryea et al.

2009), LAC, KYN and KYN-TRP ratio could be used as an indicator for endotox-

aemia in veterinary too.

General discussion

- 88 -

Figure 13: Scatterplot shows the close relationship between KYN-TRP ratio and clinical score (r =

0.66) with the data of each pig from - 30 and 180 min p.i. only. Control groups are clustered in

physiological levels of both parameters, while LPS-groups show increasing values during obser-

vation period.

Besides the degradation of the amino acid TRP, we also found decreasing levels

of ALB and TP from 120 min p.i. onwards (Renner, Kahlert et al. 2017), equal to

previous LPS-studies (Fleck, Colley et al. 1985, Goyarts and Dänicke 2006,

Kullik, Brosig et al. 2013). In PCA both variables, ALB and TP, are located along

with TRP (PC 1 ≤ 0.2, PC 2 ≥ 0.5) and oppositely to KYN, KYN-TRP ratio and TB in

the upper right quadrant (Fig. 7, dark red) which supports a significantly negative

General discussion

- 89 -

relationship between these parameters. Referring to their connections, the

measurement of TP includes all blood proteins, thus ALB, as major APP and car-

rier protein for TB, is a part of this comprehensive parameter. Additionally all

mentioned parameters have a close connection to the liver (Billing 1963, Hewett

and Roth 1993, Schröcksnadel, Wirleitner et al. 2006), which is not only a central

organ for protein metabolism but also for systemic inflammatory response and

toxin elimination (Maresca 2013). Nevertheless, toxins as DON and LPS can be

responsible for liver damage, seen in severe haemorrhages and greater relative

liver weight (Maclean, Spink et al. 1956, Mireles, Kim et al. 2005, Stanek,

Reinhardt et al. 2012) as well as in increased hepatic biochemical markers such

as TB and ASAT (Cirelli, Carey et al. 1995, Saetre, Hovig et al. 2001, Stanek,

Reinhardt et al. 2012). In our pigs both, morphological alterations and increasing

ASAT as well as hyperbilirubinaemia, that is known as a key sign of hepatic dys-

function, were observable at the end of the experiment (Renner, Kahlert et al.

2017).

Regarding to treatment, group DON_LPSjug.-CONpor. shows the severest dysfunc-

tion of the liver, seen in TB, ASAT, ALP and HAI score, albeit some of these pa-

rameters only reaching numerically higher values compared to other LPS-infused

groups. In addition, pigs of DON_LPSjug.-CONpor. respond apparently more power-

ful and effective to LPS stress compared to their counterpart, which can be de-

duced from the earlier and stronger increase of clinical signs, the lower increase

in body temperature as well as the faster decrease of WBC counts. Furthermore,

LAC and KYN-TRP ratio also suggest a more severe APR compared to the re-

maining LPS groups. These findings are in accordance with previous studies in

pigs reporting an additive or synergistic effect of DON on the LPS-induced APR

observed in liver histology and different immune parameters (Stanek, Reinhardt

et al. 2012, Gerez, Pinton et al. 2015).

In conclusion DON showed a clear priming effect (leucocytosis) in pigs. Addition-

ally we were able to proof our hypothesis that the liver differently mediates the

APR by modifications of its metabolising and detoxifying properties against an

acute LPS-intoxication for single parameters but not in summary analysis includ-

General discussion

- 90 -

ing all parameters (PCA). The APR modifications result on the one hand in a low-

er body core temperature rise, which indicates a less severe endotoxaemia.

However, the more dramatic increase of clinical signs as well as the alterations

in prognostic parameters (KYN-TRP ratio, LAC) and liver values (ASAT, ALP, TB,

ALB, TP) indicates a contradictory estimation.

Thus, further research with a longer observation period p.i. is needed to clarify

the long-term consequences of a combined toxin exposure.

Summary

- 91 -

Summary

Does chronical deoxynivalenol-feeding modulate the immune re-

sponse in endotoxaemic pigs?

Tanja Tesch (2017)

In pig farming the porcine organism is often co-exposed to different types of tox-

ins such as mycotoxins like DON and bacterial endotoxins like LPS. This can be

attributed to the widespread mycotoxin-contamination of cereal grains, one of

the main components of standard swine diets, in northern temperate climate and

the high infectious pressure in animal production by gram-negative bacteria.

Thereby the most important adverse effects of a chronic exposure to moderate

DON doses are diminished live weight gain and reduced feed intake as well as

the activation the innate immune system. Similar to DON, LPS is also regarded to

stimulate the immune system, partly occurring related molecular mechanism.

Hence, the detection of intoxications as early as possible is of crucial interest in

porcine health management to minimise economic losses in swine population.

However, knowledge about the interaction between DON and LPS, especially

with regard to the role of the liver in detoxification as well as possible modulating

effects on the APR and specific immune parameters in pigs is still limited.

Thus, the aim of the present thesis was to elucidate possible liver-related modifi-

cations of the APR in pigs co-exposed to a chronic DON-diet and an acute pre- or

post-hepatic LPS-induced endotoxaemia. Thereby we specifically focussed on

the kinetics and manifestations of clinical symptoms and different immune-

related blood parameters as well as their relationships, depending on hepatic

(portal) or systemic (jugular) LPS-infusion in combination with diet.

The experiments of the present thesis based on two feeding groups, with a total

of 44 German Landrace barrows. One group received an almost DON-free con-

trol diet (CON) while the other group was fed with a naturally DON-contaminated

diet, including 4.59 mg DON/kg feed. Pigs were fed restrictively, with 700g

feed/pig twice a day over a period of four weeks. Additionally pigs were weighed

Summary

- 92 -

once a week and blood samples were taken immediately before venous applica-

tion of an acute immune stimulus (LPS).

During this period both groups showed equal weight gain, thus nutritionally in-

duced deficiencies could be excluded. Nevertheless, DON-fed pigs showed sig-

nificantly increased leukocyte counts and thus confirmed the activation of the

immune system by chronical DON-consumption.

For the subsequent LPS-challenge pigs were surgically equipped with a perma-

nent catheter system. This facilitates the creation of different conditions focusing

on the liver functions by portal (V. splenica) or systemic (V. jugularis ext.) infu-

sions of 0.9% NaCl or 7.5 µg LPS/kg BW for 60 min as well as simultaneous

blood sampling from both localisations (V. portae hepatis, V. jugularis int.). Blood

samples were used to investigate differential blood cell counts, various inflam-

matory markers and liver values (TNF-α, KYN, TRP, TP, ALB, GLU, TB, ASAT,

LAC) as well as blood gases (pH, pO2, pCO2, BE) every 15 to 30 min.

Moreover, an intraabdominal temperature logger was inserted for steady body

core temperature measurement every 5 min. Besides core temperature meas-

urement, clinical signs and rectal temperature were also evaluated every 15 min,

starting at once with blood sampling 30 min before infusion up to 180 min p.i..

In all pigs the LPS infusion induced the APR reflected at first in severe leukopenia

and a marked increase in TNF-α. These changes in inflammatory markers were

accompanied by clear clinical signs of endotoxaemia such as tremor, cyanosis,

reddened eyes, increased respiratory rate, retching and vomiting as well as in-

creased body core and rectal temperature, independent of infusion site and diet.

However, clinical signs in DON-fed pigs showed a slower return to base levels

compared to CON-fed pigs.

The LPS induced endotoxaemia also caused a metabolic acidosis with respirato-

ry compensation indicated by decreased pH, bicarbonate, BE and pCO2 levels,

as well as later on increased LAC. Moreover, towards the end of the experiment

the response of the immune system to LPS was also reflected in increased KYN

and KYN-TRP ratio as well as in decreased TRP, TP and ALB. Additionally, an

increase in TB, ASAT and ALP as well as histological findings in HAI score indi-

cates an intense activity of the liver.

Summary

- 93 -

Regarding to these results we found significant relationships between different

inflammatory markers such as WBC counts and body temperatures as well as in

between body core and rectal temperature. Whereby these relationships were

not uniform for all conditions and site of LPS-infusion modified the magnitude of

this difference. These APR modulating effect could also be proofed in body core

temperature where DON-feeding combined with systemic LPS-infusion resulted

in a lower temperature rise compart to CON-fed pigs.

In conclusion, DON showed a clear priming effect to the immune system (leuco-

cytosis) and altered the APR resulting from a systemic immune stimulus. This

alterations result in a lower body temperature rise although blood parameters

show a more severe course of endotoxaemia. Thus, the additional measurement

of prognostic parameters (KYN-TRP ratio, LAC) seems to be a useful completion

for the clinical observation in practise to ensure the early detection of intoxica-

tions with myco- or endotoxins.

Zusammenfassung

- 94 -

Zusammenfassung

Verändert eine chronische Deoxynivalenol-Fütterung die Immunreak-

tion in endotoxämischen Schweinen?

Tanja Tesch (2017)

In der Schweinehaltung wird der Organismus der Tiere oft zeitgleich verschiede-

nen Toxinen, wie dem Mykotoxin DON und dem Bakterientoxin LPS ausgesetzt.

Dies kann der weit verbreiteten Mykotoxin-Kontamination von Getreidekörnern,

die einen Hauptanteil des Schweinefutters ausmachen, im gemäßigten Klima,

sowie dem hohen Infektionsdruck mit gramnegativen Bakterien in der Tierpro-

duktion, zugeschrieben werden. Dabei sind eine reduzierte Futterauf- und Ge-

wichtszunahme sowie die Aktivierung des angeborenen Immunsystems die

wichtigsten negativen Effekte bei einer chronischen Exposition von moderaten

DON-Konzentrationen. Vergleichbar zu DON ist auch LPS in der Lage das Im-

munsystem durch ähnliche Mechanismen zu aktivieren. Daher ist es von großer

Bedeutung für das Gesundheitsmanagement der Schweine und damit auch für

die Minimierung der wirtschaftlichen Verluste eine Intoxikation so früh wie mög-

lich festzustellen. Dennoch ist das Wissen über die Wechselwirkung zwischen

DON und LPS, vor allem hinsichtlich der Bedeutung der Leber bei der Entgiftung

sowie möglichen modulierenden Effekten auf die APR und spezifische Immunpa-

rameter in Schweinen noch sehr begrenzt.

Deshalb war das Ziel dieser Arbeit, die möglichen leberbedingten Modifikationen

der APR in Schweinen, die gleichzeitig einer chronischen DON sowie einer

akuten prä- oder post-hepatischen LPS-Endotoxämie ausgesetzt waren, näher

zu beleuchten. Dabei lag unser Fokus vor allem auf der Kinetik und Manifestati-

on der klinischen Symptomatik sowie verschiedenen immunspezifischen Blutpa-

rametern und deren Beziehungen, in Abhängigkeit von der Lokalisation der LPS-

Infusion (portal, jugular) in Kombination mit der Fütterung (DON, CON).

Die Versuche dieser Arbeit basierten zunächst auf zwei Fütterungsgruppen aus

insgesamt 44 Deutsche-Landrasse Börgen. Eine Gruppe erhielt ein nahezu

DON-freies Futter (CON), während die andere Gruppe mit einem natürlich kon-

Zusammenfassung

- 95 -

taminierten Futter, mit einer Konzentration von 4,59mg DON/kg Futter, versorgt

wurde. Die Schweine wurden über einen Zeitraum von vier Wochen zweimal

täglich mit 700g Futter/Schwein restriktiv gefüttert. Zusätzlich wurden die Tiere

wöchentlich gewogen und Blutproben wurden direkt vor der Verabreichung eines

venösen Immunstimmulus (LPS) entnommen.

Während der Fütterungs-periode zeigten beide Gruppen dieselbe Gewichtszu-

nahme, so dass ernährungsphysiologisch bedingte Mängel ausgeschlossen

werden konnten. Nichtsdestotrotz zeigten DON-gefütterte Schweine einen deutli-

chen Anstieg der Leukozytenzahlen und bestätigten damit die Aktivierung des

Immunsystems durch eine chronische DON-Aufnahme.

Für die anschließende LPS-Challenge wurden die Schweine mit einem perma-

nenten Katheter-System ausgestattet. Dies ermöglichte durch die portale (V.

splenica) oder systemische (V. jugularis ext.) Infusion von NaCl 0,9% oder 7,5µg

LPS/kg Körpergewicht über 60 min die Erschaffung verschiedener Bedingungen

im Hinblick auf die Leberfunktion, sowie die gleichzeitige Probennahme aus bei-

den Lokalisationen (V. portae hepatis, V. jugularis int.). Die alle 15 bis 30 min

entnommenen Blutproben dienten der Analyse des Differentialblutbildes, ver-

schiedener Entzündungsmarker und Leberwerten (TNF-α, KYN, TRP, TP, ALB,

GLU, TB, ASAT, LAC) sowie der Blutgasanalyse (pH, pO2, pCO2, BE).

Darüber hinaus wurde ein intraabdominaler Temperatur-logger für eine konstan-

te Messung der Körperkerntemperatur, alle 5 min, eingesetzt. Neben der Kör-

perkern- ist auch die rektale Temperatur gemessen sowie die klinische Sympto-

matik alle 15 min, beginnend mit der ersten Blutprobennahme 30 min vor Infusi-

onsbeginn bis 180 min p.i., beurteilt worden.

LPS induzierte in allen Schweinen die APR, was sich zuerst in einer starken Leu-

kopenie und einem deutlichen Anstieg von TNF-α zeigte. Diese Veränderungen

in den Entzündungsmarkern wurden begleitet von deutlichen klinischen Anzei-

chen einer Endotoxämie, wie dem Anstieg der Kern- und Rektaltemperatur, er-

höhter Atemfrequenz, Würgen, Erbrechen, gerötete Konjunktiven, Zittern und

Zyanose, in Abhängigkeit von der Infusionslokalisation und Fütterung. Allerdings

zeigen DON-gefütterte Schweine einen langsameren Rückgang der klinischen

Symptome zum Ausgangsniveau im Vergleich zu CON-gefütterten Tieren.

Zusammenfassung

- 96 -

Die LPS-induzierte Endotoxämie rief außerdem eine metabolische Azidose mit

teilweise respiratorischer Kompensation hervor, was aus erniedrigtem pH, Bikar-

bonat, BE und pCO2 sowie dem später angestiegenen LAC geschlossen werden

konnte. Des Weiteren ist die Reaktion des Immunsystems auf LPS zum Ende

des Untersuchung auch im Anstieg von KYN und KYN-TRP ratio sowie im Abfall

von TRP, TP und ALB zu erkennen. Zusätzlich lässt der Anstieg von TB, ASAT

und ALP sowie die histologischen Ergebnisse im HAI Score auf eine gesteigerte

Aktivität der Leber schließen.

Bezogen auf diese Ergebnisse haben wir deutliche Verbindungen zwischen ver-

schiedenen Entzündungsmarkern wie den Leukozyten und der Körpertempera-

tur sowie zwischen rektaler und Körperkerntemperatur gefunden. Wobei diese

Beziehungen nicht unter allen getesteten Bedingungen gleich sind und die Loka-

lisation der LPS-Infusion die Stärke der Unterschiede beeinflusst. Diese APR

modulierenden Effekte konnten auch für die Körperkerntemperatur, die bei einer

Kombination von DON-Fütterung und systemischer LPS-Applikation einen deut-

lich geringeren Anstieg zeigt als die entsprechende CON-gefütterte Gruppe, be-

stätigt werden.

Abschließend lässt sich sagen, dass DON einen klaren Priming Effekt auf das

Immunsystem (Leukozytose) hat und die APR durch einen systemischen Im-

munstimmulus ausgelöste APR verändert. Diese Veränderung resultiert in einem

geringen Anstieg der Körperkerntemperatur, obwohl die gemessenen Blutpara-

meter auf eine schwer verlaufende Endotoxämie hindeuten. Deshalb erscheint

für die Praxis eine zusätzliche Messung von prognostischen Parametern (KYN-

TRP ratio, LAC) als eine sinnvolle Ergänzung der klinischen Untersuchung um

eine frühe Feststellung einer Intoxikation mit Myko- oder Endotoxinen zu ge-

währleisten.

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