Gene expression in rabbit appendices infected with Eimeria coecicola

Veterinary Parasitology 186 (2012) 222–228

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Gene expression in rabbit appendices infected with Eimeria coecicola Mohamed A. Dkhil a,b , Mostafa A. Abdel-Maksoud a , Saleh Al-Quraishy a,∗ , Abdel-Azeem S. Abdel-Baki a,c , Frank Wunderlich a a b c

Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt Department of Zoology, Faculty of Science, Beni-Suef University, Egypt

a r t i c l e

i n f o

Article history: Received 9 August 2011 Received in revised form 3 November 2011 Accepted 7 November 2011 Keywords: Eimeria coecicola Rabbits Appendix Oligo microarrays Real time PCR

a b s t r a c t Eimeria coecicola causes intestinal coccidiosis in rabbits and, thereby, enormous economic losses in rabbit farms. Here, we investigate the final target site of E. coecicola, the appendix of rabbits, at the level of gene expression. Rabbits, orally infected with E. coecicola, begin to shed parasitic oocysts with their feces on day 5 p.i., and approximately 1.1 million oocysts are maximally shedded on day 7 p.i. At maximal shedding, the appendix has increased in size by about 2–3-folds and reveals increased hemorrhage which is associated with increases in nitrite/nitrate, malondialdehyde and catalase activity and a decrease in glutathione. Agilent 2-color oligo whole rabbit genome microarray, in combination with quantitative real-time PCR, detects 45 and 36 genes whose expression is more than 2-fold up- and down-regulated, respectively, by E. coecicola infection on day 7 p.i. The most dramatic increase by approximately 50-fold reveals the mRNA of the pro- and anti-inflammatory pleiotropic cytokine interleukin 6 (IL-6), whereas the largest decrease by approximately 13-fold is detected for mRNAs encoding for DBP, SULT3A1, CRP and glutathione-S transferase. Also, there are upand down-regulations in the expression of genes encoding diverse regions of antibodies. Our data suggest that IL-6 plays a central role in the infection of the appendix of rabbits by E. coecicola, presumably involved in both pathological injuries, host defences and healing processes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Coccidiosis is an economically important disease of farm animals including cattle, rabbits and in particular poultry. It is caused by parasitic protozoa of the genus Eimeria. These are obligate intracellular parasites predominantly invading enterocytes and are specific for both the host and the intestinal target site (Rose et al., 1992; Pakandl et al., 1993; Bhat et al., 1996). The disease spreads from one animal to the next by contact with infected feces. Typical disease symptoms are diarrhea and dehydration which can even lead to secondary septicemia and death

∗ Corresponding author. Tel.: +966 14675754. E-mail address: [email protected] (S. Al-Quraishy). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.11.031

(Vitovec and Pakandl, 1989). Worldwide the costs for control measures of coccidiosis only in cattle and poultry have been estimated to be over $800 million per year (Allen and Fetterer, 2002). Coccidiosis is treated, even prophylactically, by rather a number of diverse drugs (Greif et al., 2001; Peek, 2010). However, drug resistance phenomena and increasing concerns about an impact of these drugs on the food chain and environment are limiting their use (Peek, 2010). Reasonable alternatives are vaccines. Indeed, enormous efforts are undertaken to develop protective vaccines against different Eimeria species (Gerhold et al., 2010; Lee et al., in press), in particular E. tenella (Lee et al., in press; Del Cacho et al., 2011). Among the 800 known Eimeria species (Mehlhorn, 2001), there are eleven species which are specific for

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rabbits including Eimeria coecicola (Pakandl et al., 1993, 1995; Pakandl, 2009). Sporozoites of E. coecicola first invade the mucosa of the small intestine, especially the duodenum of the rabbits. Then, the parasites reach gut associated lymphoid tissue, including vermiform appendix, sacculus rotundus and Peyer’s patches. Eventually, the appendix is the major and final target site of E. coecicola (Pakandl et al., 2006, 2008). This target site has been previously investigated with respect to structural changes (Pakandl, 2009), but molecular changes of the host target sites in response to E. coecicola invasion have not been yet investigated to date. Here, we have used the Agilent-2-color microarray technology, in combination with quantitative real time PCR, to investigate gene expression in the appendix of the New Zealand rabbit Oryctolagus cuniculus infected with E. coecicola. 2. Materials and methods 2.1. Animals New Zealand white rabbits (O. cuniculus) at an age of 7–9 weeks and a weight of 1.5–2.5 kg are obtained from the animal facilities of King Saud University. They are maintained as described by Licois et al. (1994). Though tested for their parasite naïve statues, the rabbits are fed as a precaution on a robenidine-supplemented commercial pelleted feed until 4 days before infection, when non-supplemented pelleted feed is given (Coudert et al., 1988). 2.2. E. coecicola infections Eighteen rabbits are caged individually in autoclaved isolators at the same time. Twelve rabbits are infected with 50,000 sporulated oocysts of E. coecicola suspended in 10 ml of sterile saline by oral gavage. The oocysts are priorly collected from infected rabbits, surface-sterilized with sodium hypochlorite and washed at least four times in saline before using for oral inoculations of the experimental rabbits. Once every 24 h, the feces are collected from the individually caged rabbits and each rabbit is weighed before the bedding is re-newed to eliminate reinfection. Oocysts are counted according to Schito et al. (1996). Briefly, fecal material is suspended in 10fold volumes of 2.5% potassium dichromate, vortexed and diluted in saturated sodium chloride for oocysts flotation. Oocysts are counted in a McMaster chamber and expressed as number of oocysts per gram of wet feces.

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2.4. Biochemical determinations Nitrite/nitrate is assayed according to the technique of Berkels et al. (2004). In brief, nitrous acid is formed in acid medium and, in the presence of nitrite, the formed acid diazotizes sulphanilamide, which is coupled with N(1-naphthyl) ethylenediamine, and the resulting azo dye is measured at 540 nm. Lipid peroxidation is determined by the method of Ohkawa et al. (1979). Homogenate is suspended in 1 ml of 10% trichloroacetic acid and 1 ml of 0.67% thiobarbituric acid boiled in a water bath for 30 min. Thiobarbituric acid reactive substances are measured at 535 nm and expressed as malondialdehyde (MDA) equivalents formed. Glutathione (GSH) is measured with Ellman’s reagent (Ellman, 1959). This reagent is reduced to produce a yellow chromogen, which is directly proportional to the GSH concentration which is measured at 405 nm. Catalase (CAT) is assayed according to Aebi (1984). The reaction is started by adding H2 O2 to the homogenate for exactly 1 min, before stopping with a catalase inhibitor. The remaining H2 O2 reacts, in the presence of horse radish peroxidase, with 3,5-dichloro-2-hydroxy benzene sulphonic acid and 4-aminophenazone. The formed chromophore is inversely proportional to the CAT activity in the original sample and can be quantified at 240 nm. 2.5. Isolation of total RNA Frozen tissue is homogenized in liquid nitrogen and total RNA is isolated with Trizol (Sigma–Aldrich). Quality and integrity of RNA are determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA is quantified by measuring A260 nm on the ND-1000 Spectrophotometer (NanoDrop Technologies). 2.6. Labelling of RNA Labelling is performed as detailed in the protocol for the Two-Color Microarray-Based Gene Expression Analysis (version 5.5, part number G4140-90050). Briefly, 1 ␮g of total RNA is used for amplification and labelling using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies, Palo Alto, CA) in the presence of cyanine 3-CTP and cyanine 5-CTP (Perkin Elmer). Yields of cRNA and the dye-incorporation rate are measured with the ND-1000 Spectrophotometer (NanoDrop Technologies). 2.7. Hybridization of rabbit genome oligo microarray

2.3. Preparation of appendiceal tissue Six infected and six non-infected rabbits are euthanized with ketamine (50 mg/kg) on day 7 p.i. Appendices are then removed, cut into small pieces, and frozen in liquid nitrogen and finally stored at −80 ◦ C until use. Frozen samples are taken up in ice-cold Tris-buffer (10%, w/v) and homogenized in a PRO Scientific D-Series Benchtop homogenizer. The homogenates are used for the different biochemical determinations.

The hybridization procedure is performed according to the Two-Color Microarray-Based Gene Expression Analysis protocol (version 5.5, part number G4140-90050) using the Agilent Gene Expression Hybridization Kit (Agilent Technologies, Palo Alto, CA). Briefly, 825 ng of the corresponding Cy3- and Cy5-labelled cRNA is combined and hybridized overnight at 65 ◦ C to Agilent Whole Rabbit Genome Oligo Microarrays 4x44K containing 43,603 gene specific oligo-spots using the hybridization chamber and

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oven recommended by Agilent. After hybridization, the microarrays have been washed once with 6× SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature, which is followed by a second wash with pre-heated 0.06× SSPE buffer at 37 ◦ C containing 0.005% N-lauroylsarcosine for 1 min. Acetonitrile is used for the last washing step for 30 s. 2.8. Scanning and data analysis Fluorescence signals of the hybridized microarrays are monitored using Agilent’s Microarray Scanner System G2505B and the Scan Control Software (Agilent Technologies, Palo Alto, CA). The Agilent Feature Extraction Software (FES) version 10.2.1.3 is used to read out and process the microarray image files. For determination of differential gene expression, the FES derived output data files are further analyzed using the Rosetta Resolver® gene expression data analysis system (Rosetta Bio software). The local signal of each spot is measured inside a 300-␮m-diameter circle. The local background is determined within 40 ␮mwide rings approximately 40 ␮m distant from the signal. Then, local background is subtracted from the local signal intensity to calculate the net signal intensity and the ratio of Cy5 to Cy3. The ratios are normalized to the median of all ratios, considering only those spots with fluorescence intensities three times larger than that of the control herring sperm DNA and spotting buffer negative controls. The values represent the means of four single spots and standard deviations. 2.9. Quantitative PCR Real time PCR is performed as detailed previously (Dkhil et al., 2011). In brief, total RNA freed from DNA using the DNA free kit (Applied Biosystem, Darmstadt, Germany) is used to synthesize cDNA using QuantiTectTM Reverse QuantiTectTM SYBR® Green PCR kit (Qiagen) is applied for amplifications in the ABI Prism® 7500HT Sequence Detection System (Applied Biosystems, Darmstadt, Germany) with gene-specific QuantiTectTM primers delivered by Qiagen (Hilden, Germany). PCRs are performed and evaluated as detailed recently (Delic´ et al., 2010). Two way ANOVA is carried out in combination with Duncan’s test using a statistical package program (SPSS version 17.0). Data obtained with qRT-PCR are evaluated using Student’s t-test. 3. Results All the six rabbits infected with E. coecicola begin to shed oocysts with their feces on day 5 p.i. (Fig. 1). Maximal

Fig. 1. Course of E. coecicola infections in rabbits. Rabbits were infected with sporulated oocysts on day 0 and the fecal output of oocysts of rabbits was followed during 14 days. Values represent means ± SD (n = 6).

expulsion of approximately 1.2 million oocysts is reached on day 7 p.i., and then the output continues to decline. Approximately 200,000 oocysts were shedded on day 14 p.i. (Fig. 1). The appendix of rabbit is the final target site of E. coecicola. There is a 2–3-folds increase in the size at the maximal expulsion of oocysts on day 7 p.i. which is associated with marked hemorrhage (Fig. S1). The diameter of appendices has increased from 1.7 ± 0.1 cm to 2.8 ± 0.2 cm. Table 1 summarizes the biochemical data obtained for the appendices of rabbits infected with E. coecicola on day 7 p.i. There is an enormous increase in nitrite/nitrate in the appendices. Also, there is a slight but significant increase in the CAT activity and malondialdehyde (MDA), whereas the tissue content in glutathione (GSH) has slightly decreased (Table 1). To detect possible molecular changes induced in the appendices by E. coecicola infections, we have compared appendiceal gene expression in non-infected control rabbits vs. rabbits infected with E. coecicola on day 7 p.i. Specifically we have isolated the total RNA from individual appendices of the 6 rabbits per group, and we have pooled equal amounts of RNA before subjecting samples to Agilent 2-color microarray analysis. Among the total 43,803 oligo spots on the microarray, 2455 spots were up-regulated and 2220 spots were down-regulated (Fig. S2). In the following we concentrate only on those genes whose expressions are altered more than 2-fold. E. coecicola infections induce an upregulation of 36 genes, whereas the expression of 26 genes is reduced (Tables 2 and 3). Among the up-regulated genes, the gene encoding the pleiotropic cytokine IL-6 exhibits by far the highest upregulation by approximately 50-fold. This

Table 1 Oxidative stress biomarkers in appendix of rabbits infected with Eimeria coecicola on day 7 p.i. Group

Nitrite/nitrate (␮mol/g)

Malondialdehyde (nmol/g)

Glutathione (U/g)

Catalase (U/g)

Non-infected rabbits Infected rabbits

128.1 ± 15.2 325.3 ± 15.8*

10.5 ± 0.04 12.6 ± 0.03*

0.7 ± 0.02 0.5 ± 0.02*

1.8 ± 0.5 2.4 ± 0.2*

Values are means ± SD. * Significant difference (p ≤ 0.01).

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Table 2 Up-regulated genes in the appendix of Oryctolagus cuniculus infected with Eimeria coecicola on day 7 p.i. Gene symbol

Gene name

Fold change Inf/C

Agilent ID

Representative Public ID

Functions

IL-6

Interleukin 6

49.6

A 04 P003351

NM 001082064

MRP-8

22.10

A 04 P013127

D17405

GZMH

Macrophage migration inhibitory factor-related protein-8 Granzyme H

Cytokine plays important role in antiparasitic immune response Lymphokine regulate cell-mediated immunity

20.90

A 04 P032774

LOC100101600

Arg-1

Arginase 1

17. 6

A 04 P000176

LOC100008814

MMP3

Matrix metallopeptidase 3 Macrophage scavenger receptor 1 Keratinocyte growth factor Pre-alpha-casein

14.40

A 04 P000568

NM 001082280

14.2

A 04 P075392

NM 001082248

12.7

A 04 P013228

ENSOCUT00000006711

12.3

A 04 P000281

LOC100009257

11.8

A 04 P002474

NM 001082054

10.9

A 04 P026187

EB377955

10.8

A 04 P032757

ENSOCUT00000017957

9.7

A 04 P029782

Y13200

7.4

A 04 P013463

AF091848

6

A 04 P002913

NM 001082638

5.7 5.5

A 04 P002666 A 04 P001891

NM 001082347 NM 001082196

Calcium-binding protein expressed in multiple cell types involved in calcium signaling Aprotein, causes blood vessels to constrict, and drives blood pressure up Inflammatory mediator Activation of immune response

5.1

A 04 P013540

AF123437

Neutrophils activation

4.8

A 04 P003387

NM 001082770

Regulation of immune response

4.6

A 04 P001373

NM 001082294

Cytokine

4.6

A 04 P101909

NM 001082311

Unknown

4.4

A 04 P001244

NM 001082081

Interleukin 1 beta Neutrophil attractant/activation protein-1 Interleukin 1 alpha Caspase-10

4.4 3.1

A 04 P003216 A 04 P004968

NM 001082201 NM 001082293

A sperm-zona pellucida binding protein involved in cell adhesion Cytokine Cytokine

2.5 2.3

A 04 P002091 A 04 P000476

NM 001101684 NM 001099966

Selectin L Regulator of G-protein signaling 4 CD38 molecule

2.3 2.1

A 04 P000229 A 04 P002306

NM 001082352 NM 001082214

2.1

A 04 P002521

NM 001082683

Cox-2 CCR5

Cyclooxygenase-2 C–C chemokine receptor 5

2 2

A 04 P041862 A 04 P032682

NM 001082388 DQ017767

TLR4

Toll-like receptor 4

2

A 04 P041227

NM 001082732

IL-15

Interleukine-15

2

A 04 P075348

NM 001082216

MSR1 KGF CSN1S1

ANKRD1 nbc01c11.y1

MMP10 MYH S100A12 AGTR2 BDKRB1 IAG2 ENA-78 IL1RA CCL2

TNFAIP6

ZAN IL-1␤ NAP-1

IL-1␣ CASP10 SELL RGS4 CD38

Ankyrin repeat domain 1 (cardiac muscle) Trigeminal nerve. Unnormalized Matrix metalloproteinase 10 Myosin heavy chain isoform 2b Calgranulin C Angiotensin II receptor, type 2 Bradykinin B1 receptor Immune activation gene-2 Neutrophil-activating peptide 78 Interleukin 1 receptor antagonist Monocyte chemoattractant protein-1 Tumor necrosis factor, alpha-induced protein 6 Zonadhesin

Serine protease that is expressed by cytotoxic immune cells Metabolic enzyme reduce iNOS activity and reduce inflammation Enzyme important for wound healing, atherosclerosis and growth of tumor cells Macrophage receptor, play important role in phagocytosis A growth factor present in the epithelialization-phase of wound healing Therapeutic agent for the prophylaxis and/or treatment of cancer, autoimmune inflammatory diseases and infectious diseases Structural motifs mediate protein-protein interaction Mediates reflex arterial blood pressure decrease and vasodilatation in lower lip of the rabbit Proteolytic enzyme increase during inflammation Muscle protein

Cytokine Initiator in cell death (apoptosis and necrosis), receptor signaling and auto immune diseases Lymphocyte adhesion during inflammation Regulation of G-protein signal transduction Glycoprotein found on the surface of many immune cells play role in cell adhesion and signal transduction Inflammatory mediator Integral membrane proteins that specifically bind and respond to cytokines of the CC chemokine Cell surface receptor play role in innate immune response Cytokine

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Table 3 Down-regulated genes in the appendix of Oryctolagus cuniculus infected with Eimeria coecicola on day 7 p.i. Gene symbol

Gene name

DBP

−13.6

A 04 P000942 NM 001082204

Important role in phenotypic recovery of hemophilia

ST3A1 CRP GST

Albumin promoter (albumin D-box) binding protein Sulfotransferase C-reactive protein Glutathione S-transferase

−13.6 −12.8 −12.6

A 04 P000631 LOC100009006 A 04 P003658 NM 001082265 A 04 P003036 ENSOCUT00000011951

CYP2D24

Cytochrome P450 2D

−11.2

A 04 P013606 J02716 A 04 P001827 NM 001081997

Catalyze amine N-sulfonation Acute phase protein Catalyze a variety of reactions and play an important role in antioxidant defences Part of multicomponent electron transfer chains, called P450-containing systems Catalytic and oxidoreductive activity

A 04 P029798 LOC100009304

Myosin synthesis

A 04 P001551 NM 001082032 A 04 P040231 ENSOCUT00000010056

Lipolytic, crucial for secretion processes Part of multicomponent electron transfer chains, called P450-containing systems

A 04 P001011 NM 001082668

Complement component

A 04 P002886 NM 001082344 A 04 P013033 M35534

Albumin precursor A soluble acute phase protein that binds to bacterial lipopolysaccharide Activator of phagocytic activity of macrophages Antimicrobial peptide of macrophages

Fold change Inf/C Agilent ID

Aldo-keto reductase family −11 1, member D1 MYH Perinatal myosin heavy −11 chain Lipase, hepatic −10.6 LIPC pHPah2 Polycyclic −10 hydrocarbon-inducible cytochrome P-450d Complement component 8, −8 C8B beta polypeptide SAP Serum albumin precursor −7.8 LBP Lipopolysaccharide binding −6.3 protein −6 LOC100009136 Corticostatin-6 AKR1D1

MCP-2 KNG1 DEF3A CD1B SLC26A7

Macrophage cationic peptide 2 Kininogen Defensin NP-3a CD1b molecule Solute carrier 2, family6, member7

Representative Public ID Functions

A 04 P002316 NM 001082300

−5.7

A 04 P012774 M28884

−4.5 −4

A 04 P032766 EF472900 A 04 P000451 NM 001082298

−3.1 −2.4

A 04 P002277 NM 001089312 A 04 P014384 AY166770

is also verified by quantitative PCR (Fig. 2). Also, other macrophage-associated genes are strongly up-regulated such as MRP-8 and MSR-1 by approximately 22-fold and 14-fold, respectively. Strongly up-regulated is also the gene encoding granzyme H, which is associated with cytotoxic T cells (Edwards et al., 1999). Moreover, the mRNA-expression of the arginase Arg-1 is significantly up-regulated, which counteracts iNOS activity (Miki

A cofactor in coagulation and inflammation Antimicrobial agent found in almost all epithelial cells Anion exchange transporter Metabolism

et al., 2009). Furthermore, genes encoding ENA-78 and NAP-1 are significantly increased, which affect functions of neutrophils. Genes involved in the reorganization of extracellular matrix such as the matrix metalloproteinase 3 (MMP3) and 10 (MMP10) are highly induced in the rabbit appendices by E. coecicola. Finally, it is noteworthy that genes encoding variable regions of antibodies are up-regulated, while, other antibody-encoding genes can be also identified among the down-regulated genes (Table 4). Down-regulated genes are also those encoding proteins normally involved in cell metabolism such as ST3A1, CYP2a11, AKR1D1, and SLC26A7. Also, it is conspicuous that genes involved in innate immunity such as CRP, C8B, kininogen and defensin NP-3a are down-regulated in the appendices of rabbits infected with E. coecicola (Table 3). By far the highest down-regulation exhibits the gene DBP involved in phenotypic recovery of hemophilia (Table 3).

4. Discussion

Fig. 2. Verification of appendix gene expression by quantitative real time PCR of genes arbitrarily selected from oligo microarrays obtained from the appendix of noninfected rabbits and rabbits infected with E. coecicola on day 7 p.i. Stars indicate significant differences between infected and noninfected control animals. IL-6, interleukin-6; MRP-8, macrophage migration inhibitory factor protein 8; GRZH, granzyme H; Arg-1, arginase 1.

The target site of E. coecicola is the intestine, especially the appendix. However, a peculiarity of E. coecicola is that the early sporozoites also take an extra-intestinal route through mesenteric lymph nodes and the spleen before penetrating cells of the intestine (Renaux et al., 2001; Pakandl, 2009). Our data indicate that oral infection of the rabbit O. cuniculus with 50,000 sporulated oocysts of E. coecicola results in intracellular development and multiplication of E. coecicola in the intestine within 5 days,

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Table 4 Up- and down-regulated genes encoding diverse regions of antibodies in the appendix of rabbit infected with E. coecicola on day 7 p.i. Gene name

Agilent ID

Representative public ID

Clone mp115 VL antibody variable domain Clone mp140 VL Clone DL5554 IgM heavy chain VDJ region

5.1 3.7 −2.1

A 04 P014547 A 04 P014841 A 04 P033374

AY171847 AY171896 AF264578

Clone 2307 IgM heavy chain VDJ region Clone mp087VL antibody variable domain Ig kappa light chain Clone mp034VH antibody variable domain Clone mp191VH antibody variable domain Clone mp022VL antibody variable domain

−4.5 −4.9 −5.7 −6.1 −6.3 −6.9

A A A A A A

P014304 P014636 P012751 P015362 P015996 P014451

AF029932 AY171791 K01280 AY171685 AY175549 AY171662

A 04 P013393

AF014746

Clone N233 Ig mu heavy chain VDJ region

Fold change Inf/C

−12.1

as evidenced by the beginning of fecal output of oocysts on day 5 p.i., which reaches its maximum on day 7 p.i. The structural alterations in the rabbit’s appendices associated with E. coecicola infections have been previously described in detail by Viotec et al. (1989), mainly indicate inflammatory responses. Moreover, the cellular infiltration which occurs in the lamina propria and villous epithelium can be correlated with immunity (Rose et al., 1992). Sühwold et al. (2010) have reported that challenge infection with Eimeria bovis leads to infiltration of both CD4+ and CD8+ T cells in small intestine and large intestine segments indicating protective functions of both cell types. Our present data also suggest serious inflammatory responses. This is indicated by the increase in nitrite/nitrate suggesting oxidative damage in the appendices. Also, the increased CAT activity and the increased lipid peroxidation evidenced by the increased MDA level, as well as the lowered level of GSH indicate serious tissue inflammations in the appendices of rabbits infected with E. coecicola. In addition, the distinct changes we have detected here at the level of gene expression in the appendix of rabbits infected with E. coecicola support the view of strong inflammations in the appendices of rabbits infected with E. coecicola. For instance, the gene encoding the proinflammatory cytokine IL-1␤ is up-regulated. This in turn may explain why genes encoding phase I and phase II metabolic enzymes such as CYP2D24, ST3A1 and SLC26A7 are significantly down-regulated. Indeed, it has been previously shown in the liver of rats that IL-1␤ is able to down-regulate numerous metabolic enzymes (Kim et al., 2003, 2004). Moreover, the up-regulated expression of the metalloproteinases MMP3 and MMP10 indicates structural reorganizations in the extracellular matrix of the infected appendices presumably accompanying the hemorrhage (Stephen et al., 2009). The pleiotropic cytokine IL-6 appears to be centrally involved in the observed appendiceal alterations. Indeed, the gene encoding IL-6 is approximately 50-fold upregulated which is by far the highest expression among all up-regulated genes. IL-6 has both pro- and antiinflammatory activities (Scheller et al., 2011). The signaling mechanisms of IL-6 are rather complex (Heinrich et al., 2003; Drucker et al., 2010). It binds to the specificitydefining membrane receptor IL-6 receptor ␣ (IL-6R␣), which then recruits two membrane GP130 protein receptor molecules to activate the JAK/STAT pathway. Besides this classic IL-6 signaling, an alternative pathway exists, namely

04 04 04 04 04 04

IL-6 trans-signaling through the soluble IL-6R␣ (sIL-6R␣), which is derived by shedding of membrane-bound IL-6R␣ (Drucker et al., 2010). The circulating complex IL-6/sIL-6R␣ is able to communicate with all other cells through their membrane GP130 receptors (Knüpfer and Preiss, 2008). At least some of these cells may produce components which contribute to damage and/or healing of the appendiceal tissue infected with E. coecicola. Moreover, IL-6 is known to be important for antibody production by B cells (Gabay, 2006; Dienz et al., 2009). In accordance, we have found both up- and downregulations of genes encoding diverse regions of antibodies. Our data cannot discriminate as to whether these changes are due to B cells inherent to appendices and/or due to immigration and/or emigration of circulating B cells. At least, however, these data suggest that these antibodies may contribute to the resolution of the E. coecicola infection in the appendices. Furthermore, IL-6 is also known for its anti-inflammatory activities (Dienz et al., 2009; Scheller et al., 2011). In this sense, our data can be interpreted to show down-regulations of genes coding for components of innate immunity such as the complement component C8 and the CRP. Also, the increased expression of Arg-1 may signal a counter-regulation to reduce oxidative damage of appendiceal tissue due to increased nitrite/nitrate concentrations, since arginase is known to be activated in macrophages to decrease iNOS activity (Ehrchen et al., 2007; Daley et al., 2010). Finally, IL-6 is also known to induce wound healing (McFarland-Mancini et al., 2010). Our data suggest that infections of rabbits with E. coecicola not only induce pathology in the appendices, the final target sites of E. coecicola, but also activate, through the anti-inflammatory activities of IL-6, anti-parasite antibodies and even wound-healing mechanisms, respectively. Acknowledgment The authors appreciate the Deanship of Scientific Research at King Saud University for funding this work through research group project no. RGP-VPP-002. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vetpar.2011.11.031.

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