Intestinal and fecal biomarkers for intestinal health of poultry

Technical field of invention

Growth performance, health and welfare of domesticated birds such as broilers heavily depends on a well-functioning intestinal tract. Consequently, there is a high need to find biomarkers which are specific for gut damage and which are easily applicable in the field. The present invention discloses a set of 20 specific proteins which can be quantified in fresh fecal droppings and intestinal content of said birds and which each specifically correlate with a damaged gut.

Background art

Poultry such as broiler chickens have the lowest feed conversion of all meat-producing animals, and are therefore considered to be a relative sustainable source of animal protein, of which the production and consumption are still rising worldwide (Scanes, 2007, Cowieson and Selle, 2012). A well-functioning intestinal tract is of key importance for digestion and nutrient absorption and consequently low feed conversion, and is also crucial for health and welfare of broilers (Bailey et al., 2010). Indeed, intestinal diseases and syndromes are rather common in broilers and constitute the most important cause for treatment (Casewell et al., 2003). In poultry practice, coccidiosis is by far the most important intestinal disease (Yegani and Korver, 2008; Caly et al., 2015). Clinical diseases caused by bacterial pathogens are not common, but it is widely recognized that a variety of intestinal syndromes are affecting broiler performance, including subclinical necrotic enteritis and coccidiosis, viral enteritis, and various non-defined enteritis syndromes (Yegani and Korver, 2008). It is not evident to diagnose these subclinical entities and differentiate these from performance problems that have no infectious etiology, such as those caused by suboptimal formulated diets that not always cause intestinal damage.

The gut wall structure and morphology is a major determinant of intestinal health. Macroscopic observations of the intestinal wall at necropsy can easily be used to monitor for intestinal lesions caused by Clostridium perfringens and coccidia (Johnson and Reid, 1970), but are less clear for more subtle intestinal pathologies. Macroscopic alterations of the gut wall (such as gut wall tonus and thickness) and intestinal content (such as viscosity) can be used by experienced veterinarians as parameters for intestinal health but are to some extent subjective (Teirlynck et al., 2011). Histopathological microscopic observations are giving an accurate picture of intestinal health as villus structure, epithelial cell defects and inflammation can be scored (Yamauchi, 2002). In any case, the above mentioned intestinal health monitoring systems should be performed post-mortem and do not have high predictive diagnostic value, although in flocks animals can be sacrificed and used to monitor for disease. Quantifiable easy-to-measure biomarkers for intestinal health are still not in use in broiler chickens in practice but would be of tremendous value as a tool to monitor for subclinical intestinal entities that cause performance problems and to evaluate control methods for intestinal health, independent of whether the triggers are derived from host, nutritional or microbial factors. Epithelial damage and epithelial permeability are likely the main drivers for intestinal health problems in broiler chickens, and damage to these cells and the gut mucosa is characterized by shortening of villi, lengthening of crypts and infiltration of inflammatory cells (Teirlynck et al., 2011; Adelman et al., 2018). It has been shown that an increase of villus length and villus-to-crypt ratio is associated with improvement of growth performance (Awad et al., 2009). While an inflammatory response in the gut is essential to control and contain infections, these responses should also have an accurate transition to an anti-inflammatory state as inflammation is costing energy that affects performance (Broom and Kogut, 2018). Various systems have been developed to measure intestinal permeability, but are mainly used in experimental models (Gilani et al., 2016; Gilani et al., 2017; Wang et al., 2015). Quantification in serum or plasma of molecules that are orally administered and, because of their difference in size, can either or not cross the epithelial layer without intestinal damage and increased permeability have been shown to have value. Examples are the ratio of concentrations of lactulose and mannitol (lactulose/mannitol ratio (LMR), Gilani et al., 2017) in plasma or fluorescein isothiocyanate-dextran (FITC-dextran) in serum (Gilani et al., 2018; Kuttappan et al., 2015), after oral delivery of these compounds to the animals, with increased LMR or FITC-dextran levels indicative of high intestinal permeability. Other biomarkers for gut health have been evaluated in serum, most of them being acute phase proteins (O'Reilly and Eckersall, 2014), but these are not specific for gut damage. In addition, all the blood markers need invasive sampling, what is not preferred as diagnostic test in poultry practice. While quite some markers have been identified using transcriptomic approaches on gut tissue (Hong et al., 2014), these are also not applicable in the field. There is thus clearly a need to find an intestinal and/or fecal biomarker for intestinal health of poultry which is present in fresh fecal droppings, litter or an intestinal content sample so that it is applicable in the field. In humans, calprotectin has been used to evaluate inflammation in case of severe gut diseases, and has been shown to be reliable and specific (Canani et ai, 2008; Chang et al., 2014). However, for poultry such as chickens, no intestinal protein biomarkers for gut health are known.

Brief description of figures

Figure 1 | Mean body weight (g) (fig la), macroscopic gut appearance score (fig lb) and coccidiosis score (fig lc) per pen for control and challenge on day 26. * denotes statistical significance at p < 0.05 between control and challenged treatment. Figure 2 | Mean villus length (fig 2a), crypt depth (fig 2b), villus-to-crypt ratio (fig 2c) and T-lymphocyte infiltration (CD ₃ area%) per pen for control and challenge (fig 2d) on day 26. *** denotes statistical significance at p < 0.0001 between control and challenge group.

Description of invention

The present invention relates to a reliable, rapid and non-invasive biomarker test to diagnose gut health of poultry. With the term 'poultry' are meant domesticated birds kept by humans for their eggs, their meat or their feathers. These birds are most typically members of the superorder Galloanserae, especially the order Galliformes which includes chickens, quails and turkeys. The present invention discloses the identification of biomarkers that are indicative of intestinal pathology. The present invention further describes a gut damage model in poultry wherein a set of intestinal and/or fecal biomarkers correlate with -for example- shortening of villi and CD ₃ infiltration, the latter being markers for intestinal inflammation.

Hence, the present invention relates in first instance to a method to determine the intestinal health status of a domesticated bird comprising:

Providing a fecal sample or an intestinal content sample obtained from said domesticated bird, and

quantifying a protein, or a fragment thereof, in said fecal sample or intestinal content sample,

wherein said protein is chosen from the group consisting of: myeloid protein 1, fibronectin, annexin A5, nucleophosmin, carbonic anhydrase 2, aminopeptidase Ey, transthyretin, ovoinhibitor, apolipoprotein A-l, hemoglobin subunit beta, superoxide dismutase [Cu-Zn], alpha-actinin-4, angiotensin-converting enzyme, WD repeat-containing protein 1, mitochondrial aspartate aminotransferase, histone H2A-IV, immunoglobulin lambda chain C region, immunoglobulin lambda chain V-l region, cathepsin D and retinol-binding protein 4.

More specifically, the present invention relates in first instance to a method to determine the intestinal health status of a domesticated bird comprising: obtaining a fecal sample or an intestinal content sample of said domesticated bird, and

quantifying a protein, or a fragment thereof, in said fecal or intestinal content sample,

wherein said protein is chosen from the group consisting of: myeloid protein 1, fibronectin, annexin A5, nucleophosmin, carbonic anhydrase 2, aminopeptidase Ey, transthyretin, ovoinhibitor and apolipoprotein A-l.

The term 'intestinal health status' relates in first instance to the status of the gut wall structure and morphology which can be affected by -for example- infectious agents or a non-infectious cause such as a suboptimal formulated diet. The latter term thus mainly relates to epithelial damage and epithelial permeability which is characterized by a shortening of villi, a lengthening of crypts and an infiltration of inflammatory cells. The latter damage and inflammation markers can also be associated with a 'severe' macroscopic appearance of the gut -compared to a 'normal' appearance- when evaluated using a scoring system such as the one described by Teirlynck et al. (2011).

The term 'obtaining a fecal or intestinal content sample' refers to any means to collect of a fresh fecal dropping from said birds or intestinal content at necropsy of said birds. The terms 'intestinal content at necropsy of birds' mean a sample taken from the content present in ileum or colon after said bird is euthanized.

The terms 'quantifying a protein, or a fragment thereof, in said fecal or intestinal content sample' refers to any method known to a skilled person to quantify said proteins or fragments of in said sample. Non-limiting examples of the latter means are mass spectrometric methods (e.g. discovery and targeted proteomics, multiple reaction monitoring (MRM) assay, sequential window acquisition of all theoretical spectra assay (SWATH), ...) which require prior isolation of said proteins or fragments thereof from said sample. The latter isolation can be undertaken via protein extractions with different lysis buffers such as Sodium dodecyl sulfate (SDS)-based protein lysis buffer, Bacterial Protein extraction reagent (B-Per) or Urea-based lysis buffer with or without bead beating or other commonly used methods. Other non limiting examples of means to quantify proteins or fragments thereof are ELISA and Western Blotting which can be performed without prior protein isolation from said sample. I nstead said fecal or intestinal content sample can be diluted (10% m/v) in phosphate-buffered saline (PBS) or a 50mM Tris, 150 mM NaCI (pH 7,2) buffer with or without bead beating prior to quantification.

It should be clear that the quantification of a single protein might be sufficient to determine the intestinal health status but that also a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more proteins can be used to determine the intestinal health status of said poultry.

The protein biomarkers of the present invention which are indicative of intestinal pathology in poultry are the following:

1. Myeloid protein 1

This protein has accession number P09840 in the UniProt database (see http://www.uniprot.org/uniprot/P08940). The protein is a granule protein present in secretory granules of heterophilic granulocytes.

This protein has the following amino acid sequence (i.e. SEQ. ID N° 1; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MPALSLIALLSLVSTAFARQWEVHPPQQQGRHWAQICSGN PFNRI RGCDRYGCGNYGASR

QGKGEKHKGVDVICTDGSIVYAPFSGQLSGPIRFFHNGNAI DDGVQISGSGYCVKLVCIH

PI RYHGQIQKGQQLGRM LPMQKVFPGIVSHIHVENCDQSDPTHLLRPIPDISPPFPQQDA

HWAVVCAGNPTN EI RGCDKYGCGYFGAPRRNGKGEKHKGVDVICADGATVYAPFSGELSG

PVKFFHNGNAIDDGVQIRGSGFCVKLLCIHPI RYNGRISKGQVLGRM LPMQRVFPGI ISH

IHVENCDRSDPTSNLERGKGESEM EV 2. Fibronectin

This protein has accession number P11722 in the UniProt database (see http://www.uniprot.org/uniprot/P11722). Fibronectin (Fn) is a high molecular weight glycoprotein that consists in a soluble form in plasma and in an insoluble form as extracellular matrix (ECM) component (Pankov and Yamada, 2002). It contributes to a variety of cellular activities including wound healing. Production of fibronectin is influenced by pro- inflammatory cytokines such as I L-1 -alpha, IL-6 and TN F-alpha.

This protein has the following amino acid sequence (i.e. SEQ. ID N° 2; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

PLDQCQDSETRTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPLQAYAPLSPPTN LRL

EPNPDTGILIVSWDRSTTPGISGYRVTTAPTNGQQGSTLEEVVGADQTSCTFENLNP GVE

YNVSVYAVKDDQESI PISKTITQEVPQLTDLSFVDITDSSIGLRWTPLNASTI IGYRITV

VAAGESVPI FEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPT

DLRFTNVGPDTM RVTWTAPTSIVLSSFLVRYSPVKKEEDVAELTISPSDNVVVLTNLLPG

TEYLVRVYSVAEQHESAPLSGIQKTGLDSPTGLDFSDITANSFTVHWIAPRATITGY KIR

HHPEHGVGRPKEDRVPPSRNSITLTNLLPGTEYVVSIIAVNGREESVPLVGQQTTVS DVP

RDLEVN PTSPTSLEISWDAPAVTVRYYRITYGETGGSSPVQEFTVPGTMSRATITGLKPG

VDYTITVYAVTGRGDSPASSKPVTVTYKTEI DTPSQMQVTDVQDNSISIRWLPSSSPVTG

YRVTAVPKKGHGPTKTKNVPPDQTQVTI EGLQPTVEYMVSVYAQNQNGESLPLVETAVTN

IDRPKGLTFTEVDVDSI KIAWESPQGQVTRYRVTYSSPEDGIHELLPAPGGEEDTAELHG

LRPGSEYTI NIVAIYDDM ESLPLTGTQSTAIPPPTN LKFTQVTPTSLTVNWNAPNVRLTG

YRVRVNPKEKTGPM KEINLSPDSTSAVVSGLMVATKYEVSVYALKDSLTSRPAQGVVTTL

ENVSPPRRARVTDATETTITITWRTKTETITGFQIDAI PAASGQN PIQRTISPDVRTYTI

TGLQPGN DYKIYLYTLNENARSSPVVI DASTAIDAPSNLRFLTTTTNSLLASWQPPRAKI

TGYII RYDKPGSPAKELLPRPRPGTTEATITGLEPGTEYTIYI IAVKNNQKSEPLVGRKR

TDDLPTLITGPHPNQPDM LDVPSVDEGTPYLTNN RYDNGNGIQLPGTSGHPQTIGHQGQQ

VFFEEHGYRRPVPTTATPLRPGSRRQPPNVDEAI EI PGYQVPI IVVPSYPHSREPRRNDT

TGQEALSQTTISWRPLLESTEYIISCQPVSQDEDTLQFRVPGTSSSATLTGLTRGAT YNI

IVEALKDHRRQKVLEEVVTVGNTVSEGLNQPADDTCYDTYTGSFYSIGEEWERLSET GFK LWCQCLGFGSGHFRCDSSKWCHDNGVNYKIGEKWDRQGENGQM I DCTCLGNGKGEF

3. Annexin A5

This protein has accession number P17153 in the UniProt database (see http://www.uniprot.org/uniprot/P17153). This protein is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade.

This protein has the following amino acid sequence (i.e. SEQ ID N° 3; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MAKYTRGTVTAFSPFDARADAEALRKAM KGMGTDEETILKI LTSRN NAQRQEIASAFKTL

FGRDLVDDLKSELTGKFETLMVSLM RPARI FDAHALKHAI KGAGTN EKVLTEILASRTPA

EVQNI KQVYMQEYEANLEDKITGETSGHFQRLLVVLLQAN RDPDGRVDEALVEKDAQVLF

RAGELKWGTDEETFITI LGTRSVSHLRRVFDKYMTISGFQJ EETI DRETSGDLEKLLLAV

VKCI RSVPAYFAETLYYSM KGAGTDDDTLI RVMVSRSEIDLLDI RHEFRKN FAKSLYQM I

QKDTSGDYRKALLLLCGGDDE

4. Nucleophosmin

This protein has accession number P16039 in the UniProt database (see http://www.uniprot.org/uniprot/P16039). Nucleophosmin is a DNA binding nuclear protein which has been described as a wound-associated protein (Mellgren, 2010).

This protein has the following amino acid sequence (i.e. SEQ. ID N° 4; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MEDSAM DM ESMGPLRPQTFLFGCELKAEKEYQFKVDDEENEHQLSLRTVTLGAGAKDELH

VVEAEALDYEGNPTKVVLASLKMSVQPTVSLGGFEITPPFVLRLKCGSGPVYVSGQH LVA

LEEEPESEDEEEDTKIGNASTKRPASGGGAKTPQKKPKLSEDDEDDDEDEDDDEDDE DDL DDDEEEI KTPM KKPAREPAGKNMQKAKQNGKDSKPSTPASKTKTPDSKKDKSLTPKTPKV

PLSLEEI KAKMQASVDKGCSLPKLEPKFANYVKNCFRTEDQKVIQALWQWRQTL

5. Carbonic anhydrase 2 This protein has accession number P07630 in the UniProt database (see http://www.uniprot.org/uniprot/P07630). This enzyme causes the rapid interconversion of carbon dioxide and water to bicarbonate and protons (or vice versa), a reaction that is important for acid/base equilibrium. The enzyme is a marker for differentiation in epithelial cells. This protein has the following amino acid sequence (i.e. SEQ I D N° 5; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MSHHWGYDSHNGPAHWHEHFPIANGERQSPIAISTKAARYDPALKPLSFSYDAGTAKAIV NNGHSFNVEFDDSSDKSVLQGGALDGVYRLVQFHI HWGSCEGQGSEHTVDGVKYDAELHI VHWNVKYGKFAEALKHPDGLAVVGI FM KVGNAKPEIQKVVDALNSIQTKGKQASFTN FDP TGLLPPCRDYWTYPGSLTTPPLHECVIWHVLKEPITVSSEQMCKLRGLCFSAENEPVCRM VDNWRPCQPLKSREVRASFQ

6. Aminopeptidase Ey

This protein has accession number 057579 in the UniProt database (see http://www.uniprot.org/uniprot/057579). Aminopeptidase Ey (EC 3.4.11.20) from chicken (Gallus gallus domesticus) egg yolk is a homodimeric exopeptidase with a broad specificity for N-terminal amino acid residues at PI position of the substrate (Midorikawa et al. 1998). Aminopeptidases are members of a membrane-bound metallopeptidase family that are expressed at a high level on the brush-border membrane of enterocytes (Gal-Garber and Uni, 2000).

This protein has the following amino acid sequence (i.e. SEQ I D N° 6; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MAAGFFISKSVGIVGIVLALGAVATIIALSVVYAQEKNKSSGGSGGSDTTSTTTASTTTT

STTTASTTAAPNN PWNRWRLPTALKPESYEVTLQPFLTPDDNNMYIFKGNSSVVFLCEEA

TDLILI HSNKLNYTLQGGFHASLHAVNGSTPPTISNTWLETNTQYLVLQLAGPLQQGQHY

RLFSI FTGELADDLAGFYRSEYTEGNVTKVVATTQMQAPDARKAFPCFDEPAM KAVFTVT

MI HPSDHTAISNM PVHSTYQLQM DGQSWNVTQFDPTPRMSTYLLAFIVSQFDYVENNTGK

VQI RIWGRPAAIAEGQGEYALEKTGPILSFFERHYNTAYPLPKSDQVGLPDFNAGAM ENW

GLVTYRENSLLYDNAYSSIGNKERVVTVIAHELAHQWFGNLVTLRWWN DLWLNEGFASYV

EYLGADSAEPTWDIKDLMVLNELYTVMATDALTTSHPLTFREDEI NTPAQJSEVFDSIAY

SKGASVLRM LSDFLTEDVFKEGLQSYLHDFSYN NTVYTDLWDHLQEAVNKNSVPLPDSIG

AI M DRWTLQMGFPVVTVNTLTGSVQQSHFLLDSNSTVERPSVFNYTWIVPITWMTPSRTG

DRYWLVDVSATNSDFSVGSSTWLLLNLNVSGYFRVNYNQENWDQLLQQLSN NHQAIPVIN

RAQJ I DDAFN LARAQQVSVTLALNTTRFLSGETAYM PWQ.AALN N LQYFQLM FDRSEVFGA

MTKYIQKQVTPLFEYYRTATN NWTAIPSALM DQYNEINAISTACSYGIAECQQLATALYQ

QWRQNVSNN PIAPNLRSAIYCSAVATGGEEVWDFIWERFLEAPVVSEADKLRTALTCSTE

TWI LQRYLQYTI DPTKI RKQDATSTI NSIASNVVGQPLAWDFIRSNWRTLFGQYGGGSFS

FSRLISAVTQRFNTEFELKQLEQFKADNQDIGFGSGTRALEQALERTRTNI NWVKEN KEV

VHAWFRAETASS

7. Transthyretin

This protein has accession number P27731 in the UniProt database (see http://www.uniprot.org/uniprot/P27731). Plasma transthyretin (TTR) is a plasma protein secreted by the liver that circulates bound to retinol-binding protein 4 (RBP4) and its retinol ligand. TTR is a highly conserved protein in animal species.

This protein has the following amino acid sequence (i.e. SEQ. ID N° 7; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample): MAFHSTLLVFLAGLVFLSEAAPLVSHGSVDSKCPLMVKVLDAVRGSPAANVAVKVFKKAA

DGTWQDFATGKTTEFGEIHELTTEEQFVEGVYRVEFDTSSYWKGLGLSPFH EYADVVFTA

NDSGHRHYTIAALLSPFSYSTTAVVSDPQE

The following gray boxes indicate peptides obtained via a trypsin digest of ileal samples of SEQ ID N° 7 as is described further and represent non-limiting examples of protein fragments which can be quantified in a fecal or an intestinal content sample:

MAFHSTLLVFLAGLVFLSEAAPLVSHGSVDSKCPLMVKVLDAVRGSPAANVAVKFKK AA

DGTWQDFATGKTTEFGEIHELTTEEQFVEGVYRVEFDTSSYWKGLGLSPFH EYADVVFTA

NDSGHRHYTIAALLSPFSYSTTAVVSDPQE

8. Ovoinhibitor

This protein has accession number P10184 in the UniProt database (see http://www.uniprot.org/uniprot/P10184). Ovoinhibitor is found in egg white and is a serine proteinase inhibitor that can reduce enzymatic digestion by trypsin and chymotrypsin.

This protein has the following amino acid sequence (i.e. SEQ. ID N° 8; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MRTARQFVQVALALCCFADIAFGIEVNCSLYASGIGKDGTSWVACPRN LKPVCGTDGSTY

SN ECGICLYNREHGANVEKEYDGECRPKHVM I DCSPYLQVVRDGNTMVACPRI LKPVCGS

DSFTYDNECGICAYNAEHHTN ISKLHDGECKLEIGSVDCSKYPSTVSKDGRTLVACPRIL

SPVCGTDGFTYDNECGICAHNAEQRTHVSKKHDGKCRQEI PEI DCDQYPTRKTTGGKLLV

RCPRILLPVCGTDGFTYDNECGICAHNAQHGTEVKKSHDGRCKERSTPLDCTQYLSN TQN

GEAITACPFILQEVCGTDGVTYSNDCSLCAHNI ELGTSVAKKHDGRCREEVPELDCSKYK

TSTLKDGRQVVACTM IYDPVCATNGVTYASECTLCAHN LEQRTN LGKRKNGRCEEDITKE

HCREFQKVSPICTM EYVPHCGSDGVTYSNRCFFCNAYVQSNRTLNLVSMAAC 9. Apolipoprotein A-l

This protein has accession number P08250 in the UniProt database (see http://www.uniprot.org/uniprot/P08250). Apolipoprotein (apo) A-l is a 28 kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism.

This protein has the following amino acid sequence (i.e. SEQ ID N° 9; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MRGVLVTLAVLFLTGTQARSFWQHDEPQTPLDRIRDMVDVYLETVKASGKDAIAQFESSA

VGKQLDLKLADNLDTLSAAAAKLREDMAPYYKEVREMWLKDTEALRAELTKDLEEVK EKI

RPFLDQFSAKWTEELEQYRQRLTPVAQELKELTKQKVELMQAKLTPVAEEARDRLRG HVE

ELRKNLAPYSDELRQKLSQKLEEIREKGI PQASEYQAKVM EQLSNLREKMTPLVQEFRER

LTPYAEN LKNRLISFLDELQKSVA

The following gray boxes indicate peptides obtained via a trypsin digest of ileal samples of SEQ. ID N° 9 as is described further and represent non-limiting examples of protein fragments which can be quantified in a fecal or an intestinal content sample:

MRGVLVTLAVLFLTGTQARSFWQHDEPQTPLDRIRDMVDVYLETVKASGKDAIAQFE SSA

VGKQLDLKLADNLDTLSAAAAKLREDMAPYYKEVREMWLKDTEALRAELTKDLEEVK EKI

RPFLDQFSAKWTEELEQYRQRLTPVAQELKELTKQKVELMQAKLTPVAEEARDRLRG HVE

ELRKNLAPYSDELRQKLSQKLEEIREKGI PQASEYQAKVM EQLSNLREKMTPLVQEFRER

LTPYAEN LKNRLISFLDELQKSVA

10. Hemoglobin subunit beta

This protein has accession number P02112 in the UniProt database (see http://www.uniprot.org/uniprot D2112). The detection of hemoglobin subunit beta (HBB) in intestinal content indicates that the administered challenges induce gut leakage and endothelial damage allowing red blood cell leakage from the blood to the lumen. This protein has the following amino acid sequence (i.e. SEQ. I D N° 131; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MVHWTAEEKQLITGLWGKVNVAECGAEALARLLIVYPWTQRFFASFGNLSSPTAI LGN PM

VRAHGKKVLTSFGDAVKNLDNI KNTFSQLSELHCDKLHVDPENFRLLGDILIIVLAAHFS

KDFTPECQAAWQKLVRVVAHALARKYH

11. Superoxide dismutase

This protein has accession number P80566 in the UniProt database (see http://www.uniprot.org/uniprot/P80566). Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals to hydrogen peroxide (H2O2) and oxygen and contributes to enhanced small intestinal preservation in feline (Sun et al., 1991).

This protein has the following amino acid sequence (i.e. SEQ I D N° 132; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MATLKAVCVM KGDAPVEGVI HFQQQGSGPVKVTGKITGLSDGDHGFHVHEFGDNTNGCTS

AGAHFNPEGKQHGGPKDADRHVGDLGNVTAKGGVAEVEI EDSVISLTGPHCI IGRTMVVH

AKSDDLGRGGDNESKLTGNAGPRLACGVIGIAKC

The following gray boxes indicate peptides obtained via a trypsin digest of ileal samples of SEQ ID N° 132 as is described further and represent non-limiting examples of protein fragments which can be quantified in a fecal or an intestinal content sample:

MATLKAVCVM KGDAPVEGVI HFQQQGSGPVKVTGKITGLSDGDHGFHVHEFGDNTNGCTS

AGAHFNPEGKQHGGPKDADRHVGDLGNVTAKGGVAEVEI EDSVISLTGPHCI IGRTMVVH

AKSDDLGRGGDNESKLTGNAGPRLACGVIGIAKC 12. Alpha-actinin-4

This protein has accession number Q.90734 in the UniProt database (see http://www.uniprot.org/uniprot/Q90734). By indirect immunofluorescence, alpha-actinin-4 (ACTN4) was shown to be localized in the apical part of chicken intestinal epithelial cells (Craig and Pardo, 1979), more specifically as a component of the tight junction (zonula occludens) (Chen et al., 2006) and/or belt desmosome (zonula adherens) (Milanini et a!., 2017).

This protein has the following amino acid sequence (i.e. SEQ. I D N° 133; the gray boxes indicate peptides obtained via a trypsin digest of colon samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MVDYHSAGQPYPYGGNGPGPNGDYMAQEDDWDRDLLLDPAWEKQQRKTFTAWCNSHLRKA

GTQJENI DEDFRDGLKLM LLLEVISGERLPKPERGKM RVHKI NNVN KALDFIASKGVNVV

SIGAEEIVDGNAKMTLGM IWTN LRFAIQDISVEETSAKEGLLLWCQRKTAPYKNVNVQN

FHISWKDGLAFNALI HRHRPELIEYDKLRKDDPVTN LNNAFEVAEKYLDI PKMLDAEDIV

NTARPDEKAI MTYVSSFYHAFSGAQKAETAANRICKVLAVNQEN EHLM EDYEKLASDLLE

WIRRTIPWLEDRSPQKTIQEMQQKLEDFRDYRRVHKPPKVQEKCQLEIN FNTLQTKLRLS

NRPAFM PSEGRMVSDI NTGWQHLEQAEKGYEEWLLNEIRRLEPLDHLAEKFRQKASIHEA

WTEGKEAM LKQKDYETATLSDIKALI RKHEAFESDLAAHQDRVEQIAAIAQELN ELDYYD

SPSVNARCQ.KICDQ.WDVLGSLTHSRREALEKTEKQ.LETIDELHLEYAKRAAPFN NWM ESA

MEDLQDM FIVHTI EEIEGUAAHDQFKATLPDADREREAI LGIQREAQRIADLHSIKLSG

NNPYTSVTPQVI NSKWERVQQLVPTRDRALQDEQSRQQCN ERLRRQFAGQAN IVGPWMQT

KMEEIGRISIEM HGTLEDQLQHLKHYEQSIVDYKPNLELLEHEHQLVEEALIFDNKHTNY

TM EHI RVGWEQLLTTIARTIN EVENQI LTRDAKGISQEQMQEFRASFN HFDKDHCGALGP

EEFKACLISLGYDVENDRQGDAEFNRI MSLVDPNGSGSVTFQAFIDFMSRETTDTDTADQ

VIASFKVLAGDKNYITAEELRRELPPEQAEYCIARMAPYRGPDAAPGALDYKSFSTA LYG

ESDL 13. Angiotensin-converting enzyme

This protein has accession number Q10751 in the UniProt database (see http://www.uniprot.org/uniprot/Q10751)· Angiotensin-converting enzyme (ACE) are localized in the intestinal brush border membrane and are involved as major functional enzymes in the final stadium of protein digestion in the small intestine (Yoshioka et al., 1987).

This protein has the following amino acid sequence (i.e. SEQ. ID N° 134; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which can be quantified in a fecal or an intestinal content sample):

AKELYGNIWSNFSDPQLKKIIGSIQTLGPSNLPLDKRQQYNTILSDMDKIYSTAKVCLDN

GTCWDLEPDISDIMATSRSYKKLLYAWEGWHNAAGNPLRAKYQEFVTLSNEAYQMDG FED

TGSYWRSWYDSTTFEDDLEHLYNQLEPLYLNLHAFVRRKLYDRYGPKYINLKGPIPA HLL

GNMWAQQWNNIYDLMVPYPDKPNLDVTNTMVNQGWNATHMFRVSEEFFTSLGLLEMP PEF

WEKSMLEKPADGREVVCHASAWDFYNRKDFRIKQCTTVTMEQLFTVHHEMGHVQYYL QYK

DQPVSFRGGANPGFHEAIGDVLSLSVSTPSHLQKIGLLSSAVEDEESNINYLLKMAL EKI

AFLPFGYLIDQWRWNVFSGRTPPSRYNYDWWYLRTKYQGICAPVSRNESNFDPGAKY HIP

GNTPYIRYFVSFILQFQFHKALCQAANHTGPLHTCDIYMSKEAGAKLREVLKAGSSK SWQ

EILFNLTGTDKMDAGALLEYFSPVTTWLQEQNNKTNEVLGWPEFDWRSPIPEGYPEG IDK

IVDEAQAKEFLSEYNSTAEVVWNAYTEASWEYNTNITDHNKEVMLEKNLAMSKHTIE YGM

RARQFDPSDFQDETVTRILNKLSVLERAALPEDELKEYNTLLSDMETTYSVAKVCRE NNT

FHPLDPDLTDILATSRDYNELLFAWKGWWDASGAKIKDKYKRYVELSNKAAVLNGYT DNG

AYWRSLYETPTFEEDLERLYLQLQPLYLNLHAYVRRALYNKYGAEHISLKGPIPAHL LGN

MWAQSWSNIFDLVMPFPDATKVDATPAMKQQGWTPKMMFEESDRFFTSLGLIPMPQE FWD

KSMIEKPADGREVVCHASAWDFYNRKDFRIKQCTVVNMDDLITVHHEMGHVQYFLQY MDQ

PISFRDGANPGFHEAIGDVMALSVSTPKHLHSINLLDQVTENEESDINYLMSIALDK IAF

LPFGYLMDQWRWKVFDGRIKEDEYNQQWWNLRLKYQGLCPPVPRSEDDFDPGAKFHI PAN

VPYIRYFVSFVIQFQFHQALCKAAGHTGPLHTCDIYQSKEAGKLLGDAMKLGFSKPW PEA

MQLITGQPNMSAEALMSYFEPLMTWLVKKNTENGEVLGWPEYSWTPYAVTEFHAATD TAD

FLGMSVGTKQATAGAWVLLALALVFLITSIFLGVKLFSSRRKAFKSSSEMELK 14. WD repeat-containing protein 1

This protein has accession number 093277 in the UniProt database (see http://www.uniprot.org/uniprot/093277). WD (tryptophan-aspartate) repeat-containing protein 1 (WDR1), also called actin-interacting protein 1 (AIP1), acts as a cofactor of ADF- cofilin and facilitates actin turnover by disassembly of actin filaments (Fujibuchi et al., 2004).

This protein has the following amino acid sequence (i.e. SEQ I D N° 135; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MRM PYEI KKVFASLPQVERGVSKIIGGDPKGN NFLYTNGKCVVIRN IDNPAIADIYTEHA

HQVVVAKYAPSGFYIASGDVSGKLRIWDTTQKEHLLKYEYQPFAGKI KDLAWTEDSKRIA

VVGEGREKFGAVFLWDSGSSVGEITGHNKVINSVDIKQTRPYRLATGSDDNCAAFFE GPP

FKFKFTLSDHTRFVNCVRFSPDGN RFATASADGQJFIYDGKTGEKVCALGGGKAHDGGIY

AISWSPDSSQLLSASGDKTAKIWDVGANSVVSTFNMGSNVLDQQLGCLWQKDHLLSL SLS

GYI NYLDKN NPDKPLRVI KGHSKSIQCLTVHKNGGKSYIYSGSNDGHINYWDSDTGEN DG

FSGKGHTNQVSRMAVDEM DQLVTCSM DDTVRYTNLSKRDYSGQDAVKM DVQPKCLAVGPG

GYTVVLCIGQIVLM KDKKKCFAIDDLGYEPEAVAVHPGGGSVAVGGTDGNVRLYSIQGTS

LKSDDKTLEAKGPVTDLAYSHDGAFLAVCDAN KVVTVFSVPDGYVEHNVFYGHHAKVVCI

AWSPDNEHFASGGM DMMVYVWTVSDPETRI KIPDAHRLHHVSGLAWLDEHTLVTTSHDAS

VKEWSISYN

15. Aspartate aminotransferase, mitochondrial

This protein has accession number P00508 in the UniProt database (see http://www.uniprot.org/uniprot/P00508). Aspartate aminotransferase, mitochondrial (AATM), formerly known as glutamic-oxaloacetic transaminase, catalyzes the reaction of L- aspartate and 2-oxoglutatarate to oxaloacetate and glutamate. This mitochondrial isotype is present predominantly in liver (Kaneko et al., 2008).

This protein has the following amino acid sequence (i.e. SEQ. I D N° 136; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MALLQSRLLLSAPRRAAATARASSWWSHVEMGPPDPILGVTEAFKRDTNSKKMN LGVGAY

RDDNGKSYVLNCVRKAEAM IAAKKMDKEYLPIAGLADFTRASAELALGENSEAFKSGRYV

TVQGISGTGSLRVGAN FLQRFFKFSRDVYLPKPSWGNHTPI FRDAGLQLQAYRYYDPKTC

SLDFTGAM EDISKIPEKSII LLHACAHNPTGVDPRQEQWKELASVVKKRNLLAYFDMAYQ

GFASGDI NRDAWALRHFIEQGIDVVLSQSYAKNMGLYGERAGAFTVICRDAEEAKRVESQ

LKI LIRPMYSNPPM NGARIASLILNTPELRKEWLVEVKGMADRIISM RTQLVSN LKKEGS

SHNWQHITDQIGM FCFTGLKPEQVERLTKEFSIYMTKDGRISVAGVASSNVGYLAHAIHQ

VTK

16. Histone H2A-IV

This protein has accession number P02263 in the UniProt database (see http://www.uniprot.org/uniprot/P02263). H2A4 is a core component of nucleosomes that wrap and compact DNA into chromatin.

This protein has the following amino acid sequence (i.e. SEQ. I D N° 137; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MSGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYLAAVLEYLT

AEILELAGNAARDN KKTRI IPRHLQLAIRN DEELNKLLGKVTIAQGGVLPN IQAVLLPKK

TDSHKAKAK

17. Ig lambda chain C region

This protein has accession number P20763 in the UniProt database (see http://www.uniprot.org/uniprot/P20763). Antibodies consist of two heavy and light chains whereby birds only have one isotope of light chain, namely lambda (l). The light chain is made up by a constant, Ig lambda chain C-region (LAC), and a variable region, Ig lambda chain VI- region (LV1) (Bencina et al., 2014). This protein has the following amino acid sequence (i.e. SEQ. ID N° 138; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which can be quantified in a fecal or an intestinal content sample):

QPKVAPTITLFPPSKEELNEATKATLVCLINDFYPSPVTVDWVIDGSTRSGETTAPQRQS

NSQYMASSYLSLSASDWSSHETYTCRVTHNGTSITKTLKRSEC

18. Ig lambda chain VI region

This protein has accession number P04210 in the UniProt database (see http://www.uniprot.org/uniprot/P04210). Antibodies consist of two heavy and light chains whereby birds only have one isotope of light chain, namely lambda (l). The light chain is made up by a constant, Ig lambda chain C-region (LAC), and a variable region, Ig lambda chain VI- region (LV1) (Bencina et al., 2014).

This protein has the following amino acid sequence (i.e. SEQ ID N° 139; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which can be quantified in a fecal or an intestinal content sample):

MAWAPLLLAVLAHTSGSLVQAALTQPSSVSANPGETVKITCSGDRSYYGWYQQKAPGSAP

VTLIYDNTNRPSNIPSRFSGSKSGSTATLTITGVQADDEAVYYCGSADSSSTA

19. Cathepsin D

This protein has accession number Q.05744 in the UniProt database (see

http://www.uniprot.org/uniprot/Q05744). Cathepsin D (CATD), an aspartic proteinase, is optimally active against denatured proteins at acidic pH. CATD is expressed in lysozomes, but also exists bound to some intracellular membranes which has been detected in several different cell types (Fusek and Vetvicka, 1995).

This protein has the following amino acid sequence (i.e. SEQ ID N° 140; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MAPRGLLVLLLLALVGPCAALI RIPLTKFTSTRRM LTEVGSEIPDM NAITQ.FLKFKLGFA

DLAEPTPEI LKNYM DAQYYGEIGIGTPPQKFTVVFDTGSSN LWVPSVHCHLLDIACLLHH

KYDASKSSTYVENGTEFAIHYGTGSLSGFLSQDTVTLGN LKIKNQI FGEAVKQPGITFIA

AKFDGILGMAFPRISVDKVTPFFDNVMQQKLI EKN IFSFYLN RDPTAQPGGELLLGGTDP

KYYSGDFSWVNVTRKAYWQVHM DSVDVANGLTLCKGGCEAIVDTGTSLITGPTKEVKELQ

TAIGAKPLIKGQYVISCDKISSLPVVTLM LGGKPYQLTGEQYVFKVSAQGETICLSGFSG

LDVPPPGGPLWILGDVFIGPYYTVFDRDNDSVGFAKCV

20. Retinol-binding protein 4

This protein has accession number P41263 in the UniProt database (see

http://www.uniprot.org/uniprot/P41263). Transthyretin (TTR) is a highly conserved protein in animal species that is involved in transport of thyroid hormones and retinol bound to retinol-binding protein 4 (RET4) in the bloodstream (I ngenbleek & Bernstein, 2015). Retinol (vitamin A) is known to be essential for differentiation and proliferation of epithelial cells (Thomas et a!., 2004).

This protein has the following amino acid sequence (i.e. SEQ. I D N° 141; the gray boxes indicate peptides obtained via a trypsin digest of ileal samples as is described further and represent non-limiting examples of protein fragments which ca n be quantified in a fecal or an intestinal content sample):

MAYTWRALLLLALAFLGSSMAERDCRVSSFKVKENFDKNRYSGTWYAMAKKDPEGLFLQD

NVVAQFTVDENGQMSATAKGRVRLFN NWDVCADM IGSFTDTEDPAKFKM KYWGVASFLQK

GNDDHWVVDTDYDTYALHYSCRELN EDGTCADSYSFVFSRDPKGLPPEAQKIVRQRQI DL

CLDRKYRVIVHNGFCS

The present invention further relates to a method as described above wherein an increased level of a protein chosen from the group consisting of myeloid protein 1, fibronectin, annexin A5, nucleophosmin, carbonic anhydrase 2, transthyretin, ovoinhibitor, apolipoprotein A-l, hemoglobin subunit beta, alpha-actinin-4, histone H2A-IV and retinol-binding protein 4 present in said fecal or intestinal content sample, when compared to the level found in fecal and/or intestinal content samples of healthy control animals, is an indicator of poor intestinal health. The terms "increased levels of protein compared to the level found in healthy control animals" means at least a 2 fold increase such as a 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold ... increase compared to the level found in healthy control animals.

The present invention further relates to a method as described above wherein a decreased level of the protein aminopeptidase Ey, superoxide dismutase [Cu-Zn], angiotensin-converting enzyme, WD repeat-containing protein 1, mitochondrial aspartate aminotransferase, immunoglobulin lambda chain C region, immunoglobulin lambda chain V-l region and cathepsin D present in said fecal or an intestinal content sample, when compared to the level found in fecal and/or an intestinal content samples of healthy control animals, is an indicator of poor intestinal health. The terms "decreased levels of protein compared to the level found in healthy control animals" means at least an 2 fold decrease such as a 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold ... decrease compared to the level found in healthy control animals.

The present invention thus relates to a method as described above wherein said protein abundance is significantly differential between healthy and challenged group and/or level of protein correlates with the intestinal health as determined by measuring villus length in the duodenum of said birds, and/or, by measuring villus-to-crypt ratio in the duodenum of said birds, and/or by measuring T-lymphocyte infiltration is said villi, and/or, by scoring the macroscopic gut appearance of said birds. It is clear that said intestinal health is a measure for/correlates with performance parameters of the birds such as body weight and feed conversion ratio. The present invention further relates to a method as described above wherein said intestinal content sample is a colonic content sample and wherein said protein is chosen from the group consisting of: myeloid protein 1, fibronectin, annexin A5, nucleophosmin, carbonic anhydrase 2, aminopeptidase Ey, transthyretin, ovoinhibitor, apolipoprotein A-l, hemoglobin subunit beta, superoxide dismutase [Cu-Zn] and alpha-actinin-4. The present invention further relates to a method as described above wherein said intestinal content sample is an ileal content sample and wherein said protein is chosen from the group consisting of: aminopeptidase Ey, transthyretin, apolipoprotein A-l, superoxide dismutase [Cu-Zn], angiotensin-converting enzyme, WD repeat-containing protein 1, mitochondrial aspartate aminotransferase, histone H2A-IV, immunoglobulin lambda chain C region, immunoglobulin lambda chain V-l region, cathepsin D and retinol-binding protein 4.

More specifically, the present invention relates to a method as described above wherein said fragment of myeloid protein 1 comprises at least one of the following amino acid sequences: APFSGELSGPVK (SEQ ID N° 10), APFSGQLSGPIR (SEQ ID N° 11), FSGELSGPVK (SEQ I D N° 12), HGQIQK (SEQ I D N° 13), SDPTSNLER (SEQ ID N° 14), SGELSGPVK (SEQ I D N° 15), SGQLSGPIR (SEQ I D N° 16), VFPGI ISHI (SEQ ID N° 17), VFPGIVSH (SEQ I D N° 18), VFPGIVSHI (SEQ I D N°19); wherein said fragment of fibronectin comprises at least one of the following amino acid sequences: ATITGYK (SEQ ID N° 20), DDQESI PISK (SEQ ID N° 21) ; wherein said fragment of annexin A5 comprises at least one of the following amino acid sequences: KAM KGMGTDEETILK (SEQ I D N° 22), LLLAVVK (SEQ I D N° 23), VDEALVEK (SEQ ID N° 24); wherein said fragment of nucleophosmin comprises at least one of the following amino acid sequences: IGNASTK (SEQ I D N° 25) , TPDSK (SEQ ID N° 26), TVTLGAGAK (SEQ I D N° 27), VVLASLK (SEQ ID N° 28); wherein said fragment of ca rbonic anhydrase 2 comprises at least one of the following amino acid sequences: VGNAKPEIQK (SEQ I D N° 29), VVDALNSIQTK (SEQ ID N° 30); wherein said fragment of aminopeptidase Ey comprises at least one of the following amino acid sequences: ADNQDIGFGSGTR (SEQ ID N° 31), AIAEGQGEYALEK (SEQ I D N° 32), APVVSEADK (SEQ ID N° 33), AQII DDAFN LAR (SEQ I D N° 34), AVFTVTM IHPS (SEQ I D N° 35), AWDFIR (SEQ I D N° 36), DFIWER (SEQ I D N° 37), DFLTEDVFK (SEQ ID N° 38), DHLQEAVNK (SEQ ID N° 39), DLWDHLQEAVN K (SEQ I D N° 40), DNAYSSIGNK (SEQ ID N° 41), EAPVVSEADK (SEQ ID N° 42), EGQGEYALEK (SEQ I D N° 43), ENSLLYDNAYSSIGN K (SEQ ID N° 44), EQALER (SEQ I D N° 45), FLEAPVVSEADK (SEQ ID N° 46), FLEAPVVSEADKLR (SEQ I D N° 47), FNTEFELK (SEQ I D N° 48), GADSAETWDI K (SEQ I D N° 49), HYNTAYPLPK (SEQ I D N° 50), IAEGQGEYALEK (SEQ ID N° 51), ILSFFER (SEQ ID N° 52), IWGRPAAIAE (SEQ I D N° 53), IWGRPAAIAEGQGEY (SEQ I D N° 54), IWGRPAAIAEGQGEYALEK (SEQ ID N° 55), KQDATSTI N (SEQ ID N° 56) KQDATSTI NSI ASN VVGQPL (SEQ I D N° 57), KQDATSI NSIASNVVGQPLA (SEQ ID N° 58), LAGPLQQGQHYR (SEQ I D N° 59), LEAPVVSEADK (SEQ I D N° 60), LPTALKPESYEVTLQPF (SEQ ID N° 61), M LSDFLTEDVFK (SEQ I D N° 62), NSVPLPDSIGAI MDR (SEQ ID N° 63), PAAIAEGQGEYALEK (SEQ ID N° 64), QAI PVI NR (SEQ ID N° 65), QDATSTI NSIASNVVGQPL (SEQ I D N° 66), QNVSNNPIAPN LR (SEQ ID N° 67), SDFLTEDVFK (SEQ ID N° 68), SDQVGLPDFNAGAM ENWG (SEQ I D N° 69), SEVFDSIAYSK (SEQ I D N° 70), SLLYDNAYSSIGN K (SEQ ID N° 71), SNN HQAI PVI NR (SEQ ID N° 72), SVPLPDSIGAI M DR (SEQ ID N° 73), TDLWDHLQEAVNK (SEQ I D N° 74), TGELADDLAGFYR (SEQ ID N° 75), TGPI LSFFER (SEQ ID N° 76), TI DPTK (SEQ ID N° 77), TLFGQYGGGSFSFSR (SEQ I D N° 78), TN INWVK (SEQ ID N° 79), VNYNQENWDQLL (SEQ ID N° 80), VNYQENWDQLLQ (SEQ I D N° 81), VNYQENWDQLLQQ (SEQ ID N° 82), VVATTQMQAPDAR (SEQ ID N° 83), WRLPTAL (SEQ ID N° 84), WRLPTALKPES (SEQ ID N° 85), WRLPTALKPESYEVTLQPF (SEQ ID N° 86), YDNAYSSIGNK (SEQ ID N° 87), YLQYTI DPTK (SEQ I D N° 88), YPLPK (SEQ I D N° 89); wherein said fragment of transthyretin comprises at least one of the following amino acid sequences: AADGTWQDFATGK (SEQ ID N° 90), CPLMVK (SEQ ID N° 91), DGTWQDFATGK (SEQ I D N° 92), DVVFTAN DSGHR (SEQ I D N° 93), GLGLSPFH (SEQ ID N° 94), GLGLSPFHEY (SEQ ID N° 95), GLGLSPFHEYA (SEQ ID N° 96), GLGLSPFHEYADVVF (SEQ ID N° 97), GLGLSPFHEYADVVFTANDSGHR (SEQ ID N° 98), GSPAANVAVK (SEQ I D N° 99), GSPAANVAVKV (SEQ ID N° 100), GTWQDFATGK (SEQ ID N° 101), HYTIAALL (SEQ ID N° 102), HYTIAALLSPF (SEQ ID N° 103), HYTIAALLSPFS (SEQ ID N° 104), TTEEQFVEGVYR (SEQ ID N° 105), TTEFGEIHEL (SEQ ID N° 106), TTEFGEIHELTTEEQ (SEQ ID N° 107), TTEFGEIHELTTEEQFVEGV (SEQ ID N° 108), TTEFGEIHELTTEEQFVEGVYR (SEQ ID N° 109), TTEFGEIHELTTEEQFVEGVYRVEFDTSSYWK (SEQ ID N° 110), VEFDTSSYWK (SEQ ID N° 111), VLDAVR (SEQ ID N° 112); wherein said fragment of ovoinhibitor comprises at least one of the following amino acid sequences: EHGANVEK (SEQ I D N° 113), TLN LVSMAAC (SEQ ID N° 114), TLVACPR (SEQ I D N° 115); wherein said fragment of a polipoprotein A-l comprises at least one of the following amino acid sequences: DLEEVKEK (SEQ ID N° 116), EMWLK (SEQ I D N° 117), IRDMVDV (SEQ I D N° 118), IRPFLDQF (SEQ ID N° 119), IRPFLDQFSAK (SEQ ID N° 120), LADN LDTLSAAAAK (SEQ I D N° 121), LISFLDELQK (SEQ I D N° 122), LSQKLEEI (SEQ ID N° 123), LTPVAEEAR (SEQ ID N° 124), LTPVAQELK (SEQ ID N° 125), LTPYAENLK (SEQ ID N° 126), MTPLVQEFR (SEQ I D N° 127), QKLSQK (SEQ I D N° 128), QLDLK (SEQ I D N° 129), YKEVR (SEQ ID N° 130); wherein said fragment of hemoglobin subunit beta comprises at least one of the following amino acid sequences: KVLTSFGDAV (SEQ ID N° 142), LHVDPENF (SEQ ID N° 143), LLIVYPWTQR (SEQ I D N° 144), N LDNI K (SEQ I D N° 145), VLTSFGDAVK (SEQ ID N° 146); wherein said fragment of superoxide dismutase [Cu-Zn] comprises at least one of the following amino acid sequences: AVCVMK (SEQ ID N° 147), FQQQGSGPVK (SEQ ID N° 148),

GDAPVEGVIHFQQQGSGPVK (SEQ ID N° 149), GGVAEVEI (SEQ ID N° 150),

GGVAEVEIEDSVISLTGPH (SEQ ID N° 151), GVIGIAK (SEQ ID N° 152), HVGDLGNVTA (SEQ ID N° 153), HVGDLGNVTAK (SEQ ID N° 154), ITGLSDGDHGFHVH (SEQ ID N° 155), LACGVIGIAK (SEQ ID N° 156), LTGNAGPR (SEQ ID N° 157), SDDLGR (SEQ ID N° 158), SDDLGRGGDNESK (SEQ ID N° 159), TMVVHA (SEQ ID N° 160); wherein said fragment of alpha-actinin-4 comprises at least one of the following amino acid sequences: DAEDIVNTARDPEK (SEQ ID N° 161), TIPWLEDR (SEQ ID N° 162); wherein said fragment of angiotensin-converting enzyme comprises at least one of the following amino acid sequences: AALPEDELKEYNTLLSDMETTYSVAK (SEQ ID N° 163), ALYNK (SEQ ID N° 164), DGANPGFHEAIGDV (SEQ ID N° 165), DGANPGFHEAIGDVMA (SEQ ID N° 166), DGANPGFHEAIGDVMAL (SEQ ID N° 167), DYNELLFAWK (SEQ ID N° 168), ETPTFEEDLER (SEQ ID N° 169), EVMLEK (SEQ ID N° 170), FEESDR (SEQ ID N° 171), FFTSLGLIPMPQEFWDK (SEQ ID N° 172), GGANPGFHEAIGDVLS (SEQ ID N° 173), GLIPMPQEFWDK (SEQ ID N° 174), GLLEMPPEFWEK (SEQ ID N° 175), GPIPAHL (SEQ ID N° 176), GPIPAHLLGNMW (SEQ ID N° 177), GPIPAHLLGNMWAQQ(SEQID N° 178), GPIPAHLLGNMWAQS (SEQ ID N° 179), GYLIDQWR (SEQ ID N° 180), IIGSIQTLGPSNLPLDK (SEQ ID N° 181), IIGSIQTLGPSNLPLDKR (SEQ ID N° 182), IKEDEYNQQWWNL (SEQ ID N° 183), IYSTAK (SEQ ID N° 184), KIIGSIQTLGPSNLPLDK (SEQ ID N° 185), LLGDAMK (SEQ ID N° 186), LLYAWEGWHNAAGNPLR (SEQ ID N° 187), LSVLER (SEQ ID N° 188), MSIALDK (SEQ ID N° 189), NTILSDMDK (SEQ ID N° 190), QCTVVNMDDLITVH (SEQ ID N° 191), QFDPSDFQDETVTR (SEQ ID N° 192), QQGWTPK (SEQ ID N° 193), QQYNTILSDMDK (SEQ ID N° 194), RYVELSNK (SEQ ID N° 195), SLGLIPMPQEFWDK (SEQ ID N° 196), SLSVSTPSHLQK (SEQ ID N° 197), SLYETPTFEEDLER (SEQ ID N° 198), SMIEKPADGR (SEQ ID N° 199), SNIFDLVMPFPDATK (SEQ ID N° 200), SVSTPK (SEQ ID N° 201), SVSTPSHLQK (SEQ ID N° 202), TLGPSNLPLDK (SEQ ID N° 203), TNEVLGWPEFDWRSPIPEGYPEGIDK (SEQ ID N° 204), TSLGLIPMPQEFWDK (SEQ ID N° 205), TSLGLLEMPPEFWEK (SEQ ID N° 206), VDATPAMK (SEQ ID N° 207), VELSNK (SEQ ID N° 208), YGAEHISLK (SEQ ID N° 209), YHIPGNTPY (SEQ ID N° 210), YINLK (SEQ ID N° 211), YNELLFAWK (SEQ ID N° 212), YQGLCPPVPR (SEQ ID N° 213), YVELSNK (SEQ ID N° 214); wherein said fragment of WD repeat-containing protein 1 comprises at least one of the following amino acid sequences: IIGGDPK (SEQ ID N° 215), KVFASLPQVERGVSK (SEQ ID N° 216), VINSVDIK (SEQ ID N° 217); wherein said fragment of mitochondrial aspartate aminotransferase comprises at least one of the following amino acid sequences: GPPDPILGVTEAFK (SEQ ID N° 218), LLLSAPR (SEQ ID N° 219), MDKEYLPI (SEQ ID N° 220), MGLYGER (SEQ I D N° 221), N PTGVDPR (SEQ I D N° 222), TQLVSNLK (SEQ ID N° 223); wherein said fragment of histone H2A-IV comprises at least one of the following amino acid sequences: NDEELN K (SEQ I D N° 224), VTIAQGGVLPNIQAAVLLPK (SEQ ID N° 225); wherein said fragment of immunoglobulin lambda chain C region comprises at least one of the following amino acid sequences: DFYPSPVTVDWVI DGSTR (SEQ I D N° 226), ITLFPPSK (SEQ I D N° 227), NDFYPSPVTVDWVIDGSTR (SEQ I D N° 228), SGETTAPQR (SEQ I D N° 229), THNGTSITK (SEQ ID N° 230), TVDWVIDGSTR (SEQ I D N° 231), VAPTITLFPPSK (SEQ I D N° 232), VAPTITLFPPSKEELN (SEQ I D N° 233), VAPTITLFPPSKEELN EAT (SEQ I D N° 234), VAPTITLFPPSKEELN EATK (SEQ I D N° 235), VTHNGTSITK (SEQ ID N° 236); wherein said fragment of immunoglobulin lambda chain VI region comprises at least one of the following a mino acid sequences: ALTQPSSVSANPGETVK (SEQ I D N° 237), APGSAPVTLIYDNTNRPSN IPSR (SEQ ID N° 238), GSAPVTLIYDNTNRPSN IPSR (SEQ ID N° 239), ITCSGDR (SEQ ID N° 240), NPGETVK (SEQ I D N° 241), PSNI PSR (SEQ ID N° 242), RPSN IPSR (SEQ ID N° 243), SANPGETVK (SEQ I D N° 244), SVSANPGETVK (SEQ ID N° 245), YGWYQQK (SEQ ID N° 246); wherein said fragment of cathepsin D comprises at least one of the following amino acid sequences: DPTAQPGGELLLGGTDPK (SEQ ID N° 247), ELQTAIGAKPL (SEQ ID N° 248), ELQTAIGAKPLI (SEQ ID N° 249), FDGI LGMAFPR (SEQ ID N° 250), I PLTK (SEQ ID N° 251), QPGGELLLGGTDPK (SEQ I D N° 252), VTPFFDNVMQQK (SEQ ID N° 253); wherein said fragment of retinol-binding protein 4 comprises at least one of the following amino acid sequences: QI DLCLDR (SEQ ID N° 254) , TVDENGQMSATAK (SEQ ID N° 255).

Furthermore, the present invention relates to a method as described above wherein said domesticated bird is a broiler. The term 'broiler' refers to any chicken (Gallus gallus domesticus) that is bred and raised specifically for meat production.

Moreover, the present invention relates to a method as described above wherein said proteins or fragments thereof are quantified by using antibodies which specifically bind to said proteins or fragments thereof.

The phrase "specifically (or selectively) binds (or detects)" a protein or peptide, when referring to an antibody, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g. a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

Furthermore, the present invention relates to a method as described above wherein said antibodies are part of an Enzyme-Linked Immuno Sorbent Assay. The present invention will be further illustrated by the following, non-limiting examples.

Examples

List of abbreviations

BHI Brain Heart Infusion

BW Body weight

CD Crypt depth

CD ₃ Cluster of differentiation 3

CFU Colony forming units

DAB Di-amino-benzidine

DDA Data Dependent Acquisition

DFI Daily feed intake

DTT Dithiothreitol

DWG Daily weight gain

FCR Feed conversion ratio

HPLC-MS High performance liquid chromatography-mass spectrometry

HRP Horseradish peroxidase

LB Lysogeny Broth

MGAS Macroscopic Gut Appearance Score

MMTS Methyl methanethiosulfonate

MPDS 2 Mass prep digestion standard 2

MRS Man-Rogosa-Sharpe

MWCO Molecular weight cut-off

OTU Operational taxonomic unit

PBS Phosphate buffered saline psi Pound per square inch

TEABC Triethylammonium bicarbonate

VL Villus length

VL/CD Villus-to-crypt ratio

Materials and methods

Study design

A total of 360 day-old broilers (Ross 308) were obtained from a local hatchery and housed in floor pens on wooden shavings. Throughout the study, feed and drinking water were provided ad libitum. The broilers were randomly assigned to two treatment groups, a control and challenge group (9 pens per treatment and 20 broilers per pen). All animals were fed a commercial feed till day 12 and the feed was switched to a wheat (57.5%) based diet supplemented with 5% rye (Table 1). From day 12 to 18, all animals from the challenge group received 10 mg florfenicol and 10 mg enrofloxacin per kg body weight via the drinking water daily, to induce substantial changes in the gut microbial community. After the antibiotic treatment, 1ml of a bacterial cocktail consisting of Escherichia coli (G.78.71), Enterococcus faecalis (G.78.62), Lactobacillus salivarius (LMG22873), Lactobacillus crispatus (LMG49479), Clostridium perfringens (netB-) (D.39.61) and Ruminococcus gnavus (LMG27713) was given daily by oral gavage from day 19 till 21. Cultures of the bacteria were prepared as follows. Lysogeny Broth (LB, Oxoid,) was used for growing E. coli. Enterococcus faecalis and C. perfringens were grown in Brain Heart Infusion (BHI, Sigma) broth. Man-Rogosa-Sharpe (MRS, Oxoid) medium was used for the growth of L crispatus and L salivarius. For the growth of R. gnavus, anaerobic M2GSC medium (pH 6) as described by Miyazaki et al. (1997) was used but with 15% clarified rumen fluid instead of 30% and addition of 1 mg/ml cysteine HCI and 4 mg/ml NaHC0 ₃ after autoclaving. E. coli and Enterococcus faecalis was cultured in aerobic conditions at 37°C. In an aerobic (5% 0 ₂ ) incubator, Lactobacillus spp. were cultured. C. perfringens and R. gnavus were cultured an anaerobic chamber (gas mixture 84% N ₂ , 8% 0 ₂ en 8% H ₂ , GP[concept], Jacomex, France) at 37°C. The bacterial cells were collected by ultracentrifugation (10 000 rpm, 10 min, 20°C) and each pellet was resuspended in 100 ml anaerobic phosphate buffered saline (PBS, 1 mg/ml cysteine HCI, pH 6). The resuspended pellets were mixed and diluted with anaerobic PBS to a total volume of 1.5 I to reach a final concentration of about 10 ⁹ colony forming units (CFU)/ml for each bacterial strain (Table 2). On day 20, the animals were administered a coccidial challenge consisting of different Eimeria sp., namely 60.000 oocysts of E. acervulina and 30.000 oocysts E. maxima.

At day 26, the birds were weighed and 3 birds per pen were euthanized. The duodenal loop was sampled for histological examination and content from ileum and colon was collected for protein extraction.

Table 11 Composition and nutrient content of the wheat/ rye based broiler diet. Starter diet was given till day 11. Grower was given from day 12 to day 34. er

Wheat 55.13 57.87 Dry matter 88.45 88.38

Rye 0.00 5.00 Ash 5.11 4.79

Soy meal, crude fiber Crude protein

22.86 22.86 20.85 18.98 content < 50

Full fat soybeans 7.50 2.50 Crude fat 10.83 9.90

Animal fat 7.20 7.20 Crude fiber 2.93 2.49

Soybean oil 1.00 1.00 Carbohydrates 48.46 51.95

Premix 0.50 0.50 Starch 34.62 38.46

Lime fine 1.11 1.11 Sugars 4.77 4.53

Monocalciumphosphate 0.83 0.83 NDF 10.52 9.93

Salt 0.18 0.18 ADF 4.18 3.52

NaHCO 0.25 0.25 Calcium 0.69 0.66 L-lysine HCI 0.30 0.30 Phosphorus, total 0.57 0.54 DL-methionine 0.30 0.30 Calcium/dP poultry 0.22 0.22 L-threonine 0.10 0.10 Magnesium 0.16 0.14 Rapeseed meal < 380 2.74 0.00 Potassium 0.88 0.79

Sodium 0.15 0.15

Chloride 0.20 0.20

Base-excess (mEq/kg) 23.39 20.97 Linolic acid 2.38 1.92

Table 2 | Broilers were orally inoculated with 1 ml of a bacterial cocktail on day 19, 20 and 21 with 10 ⁶ 10 ¹⁰ CFU of Escherichia coli, Enterococcus faealis, Lactobacillus salivarius, Lactobacillus crispatus, Clostridium perfringens (netB-) and Ruminococcus gnavus

E. coli 2.11xl0 ⁹ 1.22xl0 ⁹ 2.28xl0 ⁹

Enterococcus faecalis 3.44xl0 ⁹ 2.28xl0 ¹⁰ 3.56xl0 ⁹ Lactobacillus salivarius 4.78xl0 ⁷ 1.16xl0 ⁷ 2.39xl0 ⁷ Lactobacillus crispatus 1.89xl0 ⁹ 7.78xl0 ⁷ 7.22xl0 ⁶ Clostridium perfringens lxlO ⁷ 1.06xl0 ⁷ 2.78x10 ^s Ruminococcus gnavus 2.89x10 ^s 2.78x10 ^s 3.17x10 ^s

Macroscopic scoring system

The macroscopic appearance of the gut was evaluated using a previously described scoring system (Teirlynck et al., 2011), in which in total 10 parameters were assessed and assigned 0 (absent) or 1 (present), which resulted in a total score between 0 and 10. A total score of 0 represents a normal appearance of the intestinal tract while 10 points to severe deviations from the normal appearance. The parameters are (1) 'ballooning' of the gut; (2) inflammation, cranial to the Meckel's diverticulum; (3) macroscopically visible and tangible fragile small intestine cranial to the Meckel's diverticulum; (4) loss of tonus in longitudinal cutting of the intestine cranial to the Meckel's diverticulum within the 3 seconds after incision; (5) abnormal occurrence of the intestinal content (excess mucus, orange content, gas) cranial to the Meckel's diverticulum; (6, 7, 8, 9) are identical to (2, 3, 4, 5) but caudal to the Meckel's diverticulum and (10) presence of undigested particles in the colon. A coccidiosis scoring was performed as described in Johnson & Reid (1970) which the animals were given a score for typical lesions associated with Eimeria acervulina, E. maxima and E. tenella. For each, a score was given between 0 (absent) and 4 (severe). A total coccidiosis score was calculated as the sum of the scores given for lesions caused by each individual Eimeria species. Morphological parameters

The duodenal loop was fixated in 4% formaldehyde for 24 hours, dehydrated in xylene and embedded in paraffin. Sections of 4 pm were cut using a microtome (Microme HM360, Thermo Scientific) and were processed as described by De Maesschalck et al. (2015). Morphological parameters were determined using standard light microscopy. Villus length and crypt depth in the duodenum were measured by random measurement of twelve villi per intestinal segment using Leica DM LB2 Digital and a computer based image analysis program, LAS V4.1 (Leica Application Suite V4, Germany). Also the villus-to-crypt ratio was calculated.

Immunohistochemical examination

Antigen retrieval was performed on 4 pm sections with a pressure cooker in citrate buffer (10 mM, pH 6). Slides were rinsed with washing buffer (Dako kit, K4011) and blocked with peroxidase reagent (Dako, S2023) for 5 minutes. Slides were rinsed with aqua destillata and Dako washing buffer before incubation with anti- CD ₃ primary antibodies (Dako CD ₃ , A0452) for 30 minutes at room temperature diluted 1:100 in antibody diluent (Dako, S3022). After rinsing again with washing buffer, slides were incubated with labelled polymer-HRP anti-rabbit (Envision ⁺ System-HRP, K4011) for 30 minutes at room temperature. Before adding di-amino- benzidine (DAB ⁺ ) substrate and DAB ⁺ chromogen (Dako kit, K4011) for 5 minutes, slides were rinsed 2 times with washing buffer. To stop the staining, the slides were rinsed with Aquadest, dehydrated using the Shandon Varistain-Gemini Automated Slide Stainer and counterstained with hematoxylin for 10 seconds. The slides were analyzed with Leica DM LB2 Digital and a computer based image analysis program LAS V4.1 (Leica Application Suite V4, Germany) to measure CD ₃ positive area on a total area of 3 mm ² which represents T-lymphocyte infiltration in approximately 10 villi per section. Discovery proteomics

Sample preparation

Individual colon and ileal content samples were collected and stored at -20°C before use. 500 mg was solubilized in 10 ml 2M urea, 50mM ammonium bicarbonate and homogenized by vortexing. After centrifugation (20.000xg, 15min, 4°C), the supernatant was filtered through a 0,22 pm filter unit (Merck, Germany) directly in a Vivaspin 20 with a 5 kDa MWCO filter (Sartorius, Germany) and centrifuged for lh at 4000xg. The filter was washed 3 times with 1 ml 2M urea, 50mM ammonium bicarbonate followed by centrifugation (4000xg, lOmin, 4°C). The samples were washed 3 times with 1 ml 500mM triethylammonium bicarbonate (TEABC, Sigma) to remove the urea. Subsequently, the samples were concentrated to a volume of ± 500 pi. To determine the protein concentration, a Bradford assay was performed where OD was measured at 595 nm. Approximately 50pg of proteins were reduced with ImM dithiothreitol (DTT) and incubated at 60°C for 30min, followed by alkylation for 10 min at room temperature with lOmM methyl methanethiosulfonate (MMTS). Hereafter, calcium chloride and acetonitrile were added to a final concentration of 1 mM and 5% (v/v) respectively. Finally trypsin was added in a 1:20 (trypsi protein) ratio for overnight digestion at 37°C. The samples were vacuum dried and analyzed with high performance liquid chromatography-mass spectrometry (HPLC-MS).

HPLC-MS

Peptides were dissolved in 0.1% formic acid in HPLC-grade water (buffer A) to a final concentration of lpg/pL. 100 fmol of mass prep digestion standard 2 (MPDS 2) was spiked into each sample. Data Dependent Acquisition MS analysis was performed on a TripleTOF 5600 (Sciex) fitted with a DuoSpray ion source in positive ion mode, coupled to an Eksigent NanoLC 400 HPLC system (Sciex). Peptides were separated on a microLC YMC Triart C18 column (id 300 pm, length 15 cm, particle size 3 pm) at a flow rate of 5 pL/min by means of trap-elute injection (YMC Triart C18 guard column, id 500 pm, length 5 mm, particle size 3 pm). Elution was performed using a gradient of 4-40% buffer B (0.1% formic acid, 5% DMSO in 80% ACN) over 90 min. Ion source parameters were set to 5.5 kV for the ion spray voltage, 30 psi for the curtain gas, 13 psi for the nebulizer gas and 80°C as temperature.

For DDA, a 2.25 s instrument cycle was repeated in high sensitivity mode throughout the whole gradient, consisting of a full scan MS spectrum (300-1250 m/z) with an accumulation time of 0.2 s, followed by 20 MS/MS experiments (50-1800 m/z) with 0.2 s accumulation time each, on MS precursors with charge state 2 to 5+ exceeding a 500 cps threshold. Rolling collision energy was used as suggested by the manufacturer and former target ions were excluded for 10 s.

Database searching

The *.wiff files generated during LC-MS/MS analysis were imported into the Progenesis QJ for Proteomics software (Non-linear Dynamics). The different samples were aligned based on retention time and m/z of reoccurring features to enable relative quantification. After subsequent peak picking, a merged *.mgf file was exported from the software and searched for identifications with MASCOT Daemon (Matrix Science, version 2.5.1) against a chicken database (reviewed protein database downloaded from Swissprot, January 2016) supplemented with the cRAP database (laboratory proteins and dust/contact proteins http://www.thegpm.org/crap/) and the internal standard. Maximum peptide mass tolerance and fragment mass tolerance were set to 10 ppm and 0.1 Da respectively. Additionally, methylthio on cysteine was set as a fixed modification and deamidation of asparagine and/or glutamine and oxidation of methionine were set as variable modifications. Enzyme specificity was set to trypsin with a maximum of one missed cleavage. The identifications were exported from MASCOT Daemon with a 5% false discovery rate (*.xml format) and imported into Progenesis Ql for Proteomics.

Statistical analysis

Statistical analysis was performed with Graphpad Prism (v.5). To evaluate whether the data is normally distributed, a Kolmogorov-Smirnov test was performed. In case of a normal distribution, comparison of the data was performed with an independent samples t-test. Otherwise, the non-parametric Mann-Whitney test was performed. A p-value of <0.05 was considered statistically significant. The statistical coherence between different parameters was evaluated via correlation analysis. Results

Performance parameters

Body weight (BW), daily weight gain (DWG), daily feed intake (DFI) and feed conversion ratio (FCR) were measured during different time periods. Significant differences between treatment and control groups were seen at day 26, 35 and 41, but not at the age of 12 days (Table 3).

Table 3 | Mean ± standard deviation of body weight (BW), daily weight gain (DWG), daily feed intake (DFI) and feed conversion ratio (FCR) measured during different time periods for the control and challenge group. Significant differences (p < 0.05) are shown in bold.

Control Challenge

Time

parameters Mean ± SD Mean ± SD p-value period

BW (g) 290 ± 13 295 ± 11 0.485 DWG (g) 19,6 ± 1 20,2 ± 1 0.342

D1-D12

DFI (g) 24 ± 1 25 ± 1 0.614 FCR 1.25 ± 0.06 1.23 ± 0.05 0.321 BW (g) 1375 ± 58 1195 ± 46 < 0.001 DWG (g) 78 ± 4 64 ± 4 < 0.001

D12-D26

DFI (g) 116 ± 6 108 ± 7 0.014 FCR 1.50 ± 0.07 1.69 ± 0.18 0.004 BW (g) 2345 ± 174 2153 ± 129 0.003 DWG (g) 106 ± 10 105 ± 6 0.857

D26-D35

DFI (g) 185 ± 10 203 ± 15 0.016 FCR 1.76 ± 0.14 1.94 ± 0.20 0.040 BW (g) 3095 ± 164 2850 ± 176 0.001

D35-D41 DWG (g) 121 ± 17 114 ± 21 0.276

DFI (g) 161 ± 14 166 ± 10 0.427 FCR 1.35 ±0.14 1.53 ±0.45 0.041

DWG (g) 86 ±5 76 ±4 < 0.001 D12-D35 DFI (g) 136 ±6 135 ±8 0.811

FCR 1.59 ±0.08 1.79 ±0.18 0.002

DWG (g) 91 ±4 82 ±5 < 0.001 D12-D41 DFI (g) 139 ±7 140 ±7 0.864

FCR 1.52 ±0.04 1.72 ±0.13 < 0.001

Macroscopic scoring

The appearance of the gut and a coccidiosis score were given to 27 birds per treatment. A lower body weight was observed at day 26 (p = 0.0001) for broilers which received challenge treatment. The score forthe macroscopic appearance of the gut and the total coccidiosis score were higher in the challenged group at day 26 (p < 0.001) (Figure 1, Table 4).

Table 4| Mean ± standard deviation of body weight (BW), macroscopic gut appearance score (MGAS) and coccidiosis score (CS) for the control (n = 27) and challenge group (n = 27) at day 26. Significant differences (p < 0.05) are shown in bold.

Control Challenge

Timepoint parameters Mean ± SD Mean ± SD p-value

BW (g) 1375 ±157.4 1187 ±170.1 p = 0.0001

D26 MGAS 0.9 ±0.7 3.1 ± 1.1 p < 0.001

CS 0.8 ±0.7 3.5 ±1.7 p < 0.001

Intestinal morphology and immunohistochemistry

A significant shorter villus length, an increased crypt depth, a lower villus-to-crypt ratio and a higher inflammation level in duodenal sections on day 26 (p < 0.0001) were detected in the gut of animals from the treatment group as compared to the control group (Table 5).

Table 5 | Mean ± standard deviation of villus length (VL), crypt depth (CD), villus-to-crypt ratio (VL/CD) and T-lymphocyte infiltration (CD ₃ area%) on day 26 for control (n = 27) and challenge group (n = 27). For all evaluated parameters, statistical significance was p < 0.0001.

Control Challenge

Timepoint parameters Mean ± SD Mean ± SD

VL (pm) 2035.7 ± 134.6 1369.9 ± 158.7

D26 CD (pm) 190.1 ± 15.43 365.7 ± 31.41

VL/CD 11.03 ± 1.03 3.85 ± 0.63

CD ₃ area% 7.88 ± 1.35 9.54 ± 2.71

Correlations

Pearson r has a value between -1 (total negative correlation) and +1 (total positive correlation). In case of a positive correlation, one parameter increases as the other parameter increases and vice versa. When one parameter decreases and the other one increases, there is a negative correlation. On day 26, all macroscopic (macroscopic gut appearance score, coccidiosis score and body weight) and histological parameters (villus length, crypt depth, villus-to-crypt ratio and T-lymphocyte infiltration) correlate with one another (Table 6).

Table 6 | Pearson correlation coefficient between macroscopic gut appearance score (MGAS), coccidiosis score (CS), body weight (BW), villus length (VL), crypt depth (CD), villus- to-crypt ratio (VL/CD) and T-lymphocyte infiltration (CD ₃ area%). Correlation coefficients with a statistical significance of p < 0.05 are shown.

MGAS CS BW VL CD VL/CD CD ₃ area% MGAS

CS 0.0905

BW -0.6836 -0.5974

VL -0.8400 -0.8572 0.7400

CD 0.8220 0.8045 -0.7546 -0.9073

VL/CD -0.8507 -0.8599 0.7303 0.9570 -0.9773

CD3 area% 0.8559 0.8177 -0.770 -0.9028 0.8959 -0.8979

Discovery proteomics 1.

Using MASCOT Daemon (Matrix Science, version 2.5.1) against a chicken database (reviewed protein database downloaded from Swissprot, January 2016) supplemented with the cRAP database (laboratory proteins and dust/contact proteins http://www.thegpm.org/crap/), 157 proteins were identified for colon. In theory, a good gut health biomarker should relate with one or more histological parameters since measurement of villus length and inflammation level are used as standard measurements in the evaluation of intestinal health. Also correlation with the macroscopic gut appearance score was evaluated. It is noted that proteins of which the colonic concentration has a negative correlation with the villus length, inverse correlation was seen with crypt depth, CD ₃ area% and macroscopic gut appearance score. Only correlations with a statistical significance of p < 0.1 are shown (Table 7).

Table 7 | Pearson correlation coefficient between the abundance of the protein in colon content and villus length (VL), crypt depth (CD), villus-to-crypt ratio (VL/CD) and T- lymphocyte infiltration (CD ₃ area%) and macroscopic gut appearance score (MGAS) on day 26. Correlation coefficients with a statistical significance of p < 0.1 are shown.

Accession

VL CD ₃ area% MGAS number

P08940 Myeloid protein 1 -0,7283 0,5466 0,5668

P11722 Fibronectin -0,8114 0,6515 0,6519

P17153 Annexin A5 -0,5746 P16039 Nucleophosmin -0,5263 0,7196

P07630 Carbonic anhyd rase 2 -0,5899 0,5726

057579 Aminopeptidase Ey 0,5462 -0,7177

P27731 Transthyretin -0,5778 0,6065

P10184 Ovoinhibitor -0.4852

P08250 Apolipoprotein A-l -0,5066

Discovery proteomics 2

Using MASCOT Daemon (Matrix Science, version 2.5.1) against a chicken database (reviewed protein database downloaded from Swissprot, January 2016) supplemented with the cRAP database (laboratory proteins and dust/contact proteins http://www.thegpm.org/crap/), 157 and 181 proteins were identified for colon and ileum respectively whereby significant differential proteins between control and challenged birds were selected (p < 0.05). In broilers from the challenge group, the following proteins showed a significantly higher abundance compared to control animals in colonic content (p < 0.05): alpha-actinin-4 (ACTN4), annexin A5 (ANXA5), apolipoprotein A-l (APOA1), fibronectin (FINC), hemoglobin subunit beta (HBB), myeloid protein 1 (MIM1), nucleophosmin (NPM), ovoinhibitor (IOV7) and transthyretin (TTR). Both in colonic and ileal content, superoxide dismutase [Cu-Zn] (SOD) showed a decreased abundance compared to control animals (p < 0.05). Angiotensin-converting enzyme (ACE), mitochondrial aspartate aminotransferase (AATM), cathepsin D (CATD), Ig lambda chain C region (LAC), Ig lambda chain V-l region (LV1), TTR and WD repeat-containing protein 1 (WDR1) showed a lower abundance in challenged birds (p < 0.05) in ileal samples. Following proteins were more abundant (p < 0.05): APOA1, histone H2A-IV (H2A4) and retinol-binding protein 4 (RET4) (Table 8 and Table 9). Table 8: Significantly different proteins between control and challenge group in colonic content.

Accession

Protein name Abbreviation P-value Highest mean number

Q90734 Alpha-actinin 4 ACTN4 0.0385 Challenge

P17153 Annexin A5 ANXA5 0.0266 Challenge

P08250 Apolipoprotein A-l APOA1 0.0277 Challenge

P11722 Fibronectin FINC 0.0106 Challenge

P02112 Hemoglobin subunit beta HBB 0.0158 Challenge

P08940 Myeloid protein 1 MIM1 0.0008 Challenge

P16039 Nucleophosmin NPM 0.0071 Challenge

P10184 Ovoinhibitor IOV7 0.0254 Challenge

P80566 Superoxide dismutase [Cu-Zn] SOD 0.0287 Control

P27731 Transthyretin TTR 0.0317 Challenge

Proteomics using high performance liquid chromatography-mass spectrometry (HPLC-MS) was performed on colonic content of animals from the control (n = 9) and challenged (n = 9) group at day 26. This resulted in significant differential proteins (p < 0.05) with a higher normalized abundance of 9 proteins and a decrease of superoxide dismutase [Cu-Zn] in challenged birds.

Table 9: Significantly different proteins between control and challenge group in ileal content.

Accession

Protein name Abbreviation P-value Highest mean number

057579 Aminopeptidase Ey AMPN 0.0012 Control

Q10751 Angiotensin-converting enzyme ACE 0.0006 Control

P08250 Apolipoprotein A-l APOA1 0.0364 Challenge

Aspartate aminotransferase,

P00508 A ATM 0.0067 Control mitochondrial

Q05744 Cathepsin D CATD 0.0203 Control P02263 Histone H2A-IV H2A4 0.0079 Challenge

P20763 Ig lambda chain C region LAC 0.0155 Control

P04210 Ig lambda chain V-l region LV1 0.0370 Control

P41263 Retinol-binding protein 4 RET4 0.0399 Challenge

P80566 Superoxide dismutase [Cu-Zn] SOD 0.0004 Control

P27731 Transthyretin TTR 0.0091 Control

093277 WD repeat-containing protein 1 WDR1 0.0027 Control Proteomics using high performance liquid chromatography-mass spectrometry (HPLC-MS) was performed on ileal content of animals from the control (n = 9) and challenged (n = 9) group at day 26. This resulted in 12 significant differential proteins (p < 0.05) with a higher normalized abundance of apolipoprotein A-l (APOA1), histone H2A-IV (H2A4) and retinol-binding protein 4 (RET4) and decrease in normalized abundance for the other 9 proteins in challenged birds.

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