Martijn J Wilmer

Leo A J Kluijtmans

Thea J Van Der Velden

Peter H Willems

Peter G Scheffer

Rosalinde Masereeuw

Leo A Monnens

Lambertus P Van Den Heuvel

Elena N Levtchenko
email: elena.levtchenko@uz.kuleuven.ac.

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

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Introduction

Since cysteamine therapy has become available for patients with nephropathic cystinosis (MIM219800) in the early 80's, quality of life for these patients has greatly improved [START_REF] Yudkoff | Effects of cysteamine therapy in nephropathic cystinosis[END_REF][START_REF] Gahl | Early oral cysteamine therapy for nephropathic cystinosis[END_REF].

The most frequent and most severe form, infantile cystinosis, is characterized by the development of renal Fanconi syndrome in the first year of life and leads to end stage renal disease in the first decade of life when untreated [START_REF] Gahl | Cystinosis: a Disorder of Lysosomal Membrane Transport[END_REF]. Mutations in the CTNS gene, encoding for lysosomal cystine transporter cystinosin are the cause of cystinosis [START_REF] Town | A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis[END_REF]. Lysosomal accumulation of cystine, which is the hallmark of this autosomal recessive disorder, can be depleted by the amino-thiol cysteamine [START_REF] Gahl | Cysteamine depletes cystinotic leucocyte granular fractions of cystine by the mechanism of disulphide interchange[END_REF]. Although treatment with cysteamine substantially decreases intracellular cystine accumulation, renal Fanconi syndrome is not cured, but end stage renal disease can be postponed in the majority of the patients. On the other hand, recent findings in a ctns -/-mouse model suggest that cystine accumulation itself is not sufficient for the development of renal aberrations [START_REF] Cherqui | Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis[END_REF]. Several mechanisms have been postulated to link lysosomal cystine accumulation in cystinosis with renal tubular defects, such as impaired ATP synthesis [START_REF] Coor | Role of Adenosine-Triphosphate (Atp) and Nak Atpase in the Inhibition of Proximal Tubule Transport with Intracellular Cystine Loading[END_REF][START_REF] Levtchenko | Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts[END_REF], involvement of altered glutathione (GSH) metabolism [START_REF] Chol | Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines[END_REF][START_REF] Levtchenko | Altered status of glutathione and its metabolites in eystinotic cells[END_REF] and increased apoptosis rate [START_REF] Park | Increased apoptosis in cystinotic fibroblasts and renal proximal tubule epithelial cells results from cysteinylation of protein kinase Cdelta[END_REF][START_REF] Sansanwal | Caspase-4 may play a role in loss of proximal tubules and renal injury in nephropathic cystinosis[END_REF].

Decreased levels of total GSH in cystinotic fibroblasts during exponential growth were first reported by Chol et al. [START_REF] Chol | Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines[END_REF]. This finding was confirmed in primary proximal tubular cells derived from urine [START_REF] Laube | Glutathione depletion and increased apoptosis rate in human cystinotic proximal tubular cells[END_REF]. In contrast, normal total GSH levels but increased oxidized GSH (GSSG) levels have been reported in cystinotic fibroblasts grown to confluence, in polymorphonuclear cells and in proximal tubular cells immortalized with HPV E6/E7 [START_REF] Levtchenko | Altered status of glutathione and its metabolites in eystinotic cells[END_REF][START_REF] Mannucci | Impaired activity of the gamma-glutamyl cycle in nephropathic cystinosis fibroblasts[END_REF][START_REF] Wilmer | Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells[END_REF]. Together with the finding of elevated urinary 5-oxoproline (pyroglutamic acid), a precursor for GSH synthesis, these data suggested that impaired lysosomal cystine efflux in cystinosis affects GSH metabolism, likely via impairment of γ-glutamyl-cycle [START_REF] Rizzo | Pyroglutamic aciduria and nephropathic cystinosis[END_REF].

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Alterations in GSSG/2GSH ratio affect intracellular metabolic functions and point towards increased intracellular oxidation [START_REF] Schafer | Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple, Free Radic[END_REF].

In addition to alterations in ATP-dependent GSH synthesis, decreased intracellular ATP levels have been reported in cells loaded with cystine dimethylester (CDME) [START_REF] Coor | Role of Adenosine-Triphosphate (Atp) and Nak Atpase in the Inhibition of Proximal Tubule Transport with Intracellular Cystine Loading[END_REF]. Coor et al. used this model to postulate that decreased ATP synthesis accounts for decreased sodium gradient and, consequently, influences tubular reabsorption in cystinosis. Despite the limitations of the CDME model [START_REF] Wilmer | Cystine Dimethylester Model of Cystinosis: Still Reliable?[END_REF], this hypothesis is still pending, since decreased ATP levels in cystinotic fibroblasts and proximal tubule cells were reported [START_REF] Levtchenko | Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts[END_REF][START_REF] Laube | Glutathione depletion and increased apoptosis rate in human cystinotic proximal tubular cells[END_REF]. More recently, decreased ATP generation capacity was shown in cystinotic renal proximal tubular cells [START_REF] Sansanwal | Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis[END_REF].

Despite extensive research, definite conclusions whether alterations in GSH and/or ATP status are involved the pathogenesis of cystinosis are lacking. In addition, the question remains why cysteamine treatment postpones the progression of renal disease and has no curative effect on Fanconi syndrome, despite the decrease in cystine levels. Likely, the antioxidative effect of cysteamine plays a role in this process, as cysteamine was demonstrated to increase GSH levels in mouse fibroblasts [START_REF] Djurhuus | Growth state dependent increase of glutathione by homocysteine and other thiols, and homocysteine formation in glutathione depleted mouse cell lines[END_REF]. This study focuses for the first time on the influence of cysteamine on both GSH and ATP metabolism in vitro using a recently developed conditionally immortalized proximal tubular epithelial cell line (ciPTEC) [START_REF] Racusen | Renal Proximal Tubular Epithelium from Patients with Nephropathic Cystinosis -Immortalized Cell-Lines As In-Vitro Model Systems[END_REF][START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF].

Here, 4 control and 10 cystinotic ciPTEC lines with different mutations in the CTNS gene were developed from healthy controls and patients, using identical methodology. This allowed a valuable comparison of their metabolic status in presence and absence of cysteamine to improve our insight in the mechanism of cysteamine efficacy in cystinosis.

Materials and Methods

Cell culture

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Primary cell lines were cultured from urine of 4 healthy controls (age 60-152 months old) and 10 patients with cystinosis (age 11-209 months old) after approval of the study design by the Institutional Review Board and obtaining written informed consent by the parents of all subjects as described previously [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF]. Diagnosis of cystinosis was made by measuring elevated intracellular cystine levels in polymorphonuclear granulocytes and confirmed by molecular analysis of the CTNS gene (table 1). Control cell lines were obtained from pediatric healthy volunteers with no clinical history of renal disease, nor with any other chronic disease. To maintain cell proliferation, primary cell lines were transfected with SV40T ts A58 (SV40T) and hTERT (human telomerase reverse transcriptase) followed by subcloning procedures to obtain homozygous cell populations [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF][START_REF] Bodnar | Extension of life-span by introduction of telomerase into normal human cells[END_REF][START_REF] Stamps | Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct[END_REF]. Proximal tubular origin of all tested cell lines was confirmed as described previously [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF] by demonstrating presence of aminopeptidase N, aquaporin1, P-glycoprotein and dipeptidyl peptidase IV using Western blotting. Additionally, sodium-dependent phosphate transport and alkaline phosphatase activity were analysed. Of each donor, one subclone was selected on the basis of expression pattern and activity of the proximal tubular characteristics and used for further experiments, unless mentioned otherwise. In total, subcloned ciPTEC were used from 10 cystinotic patients and 4 healthy controls. Routinely, cells were maintained at permissive temperature of 33°C (proliferating cells). To decrease expression of SV40T, cells were transferred to 37°C and allowed to mature for up to 10 days (matured cells).

Cell proliferation

To compare cell proliferation rate between control (n=4) and cystinotic (n=6) cell lines, cells were counted at several stages up to 10 days of maturation at 37°C. Data are expressed as percentage increase in cell number when transferred from 33°C to 37°C. Additionally, total intracellular protein content was measured using Biorad Protein assay (Biorad Laboratories, Germany) during maturation of cells cultured in 24 well plates.

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Protein synthesis was determined at day 10 of maturation by measuring

Albumin uptake in ciPTEC

Albumin uptake was performed as previously described [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF] by analysing BSA-FITC uptake by flow cytometry. Briefly, ciPTEC of controls (n=4) and cystinotic patients (n=7) were incubated for 30 min with 50 µg/ml BSA-FITC. Mean fluorescence intensity was presented as mean +/-SEM. To confirm that albumin uptake was megalin dependent, we performed receptor associated protein (RAP) inhibition assays on BSA-FITC uptake. RAP was kindly provided to us by Dr. M. Nielsen (University of Aarhus, Denmark).

Thiols and disulfide determination

Matured ciPTEC were harvested using trypsine/EDTA, washed twice in PBS and pellets were shock frozen in liquid nitrogen and stored at -80°C until further processing. Total intracellular content of cysteine, cystine, GSH and GSSG was measured using HPLC as described before [START_REF] Levtchenko | Altered status of glutathione and its metabolites in eystinotic cells[END_REF]. Cystine levels were measured in all control (n=4) and cystinotic (n=10) ciPTEC after

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maturation for 10 days at 37°C. To test the effect of cysteamine on intracellular cystine levels, we performed a series of experiments varying its concentration (0.2; 1; 2 mM) and time of incubation (0.5; 1; 2; 4; 8; 16; 24; 48 hrs). To study the effect of long-term cysteamine treatment (1 mM) on intracellular thiol and disulfide levels, control (n=4) and cystinotic (n=7) ciPTEC were incubated in the presence of 1 mM cysteamine for 48 hours during which cysteamine supplemented culture medium was refreshed every 6 hours. Data are expressed as nmol thiol/mg protein (+/-SEM). Furthermore, correlations between intracellular cystine and GSSG levels were determined.

Redox status calculation

Redox status (E h ) of GSSG/2GSH pool was calculated using the Nernst equation according to Schafer and Buettner [START_REF] Schafer | Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple, Free Radic[END_REF]:

(E h (mV) = E 0 -RT/nF log ([GSH] 2 /[GSSG]),
where E 0 for pH 7.4 = -264 mV; RT/nF = 30 and concentrations for GSH and GSSG in M are calculated assuming that 1 mg protein corresponds to a cellular volume of 5 µl [START_REF] L'allemain | Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport[END_REF].

Determination of ROS and oxidation status

Possible alterations in reactive oxygen species (ROS) formation in cystinotic ciPTEC were analysed using the probes 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H 2 DCFDA), and hydroethidine (HEt) [START_REF] Verkaart | Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency[END_REF]. Measurements were performed in matured control (n=4) and cystinotic (n=10) ciPTEC.

The production of ROS using CM-H 2 DCFDA was detected in ciPTEC using a 96 well assay [START_REF] Scharstuhl | Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation[END_REF] Alternatively, matured ciPTEC were loaded with HEt (10 µM) in HEPES-Tris buffer for 15 min in 24 well plates in presence or absence of the positive control rotenone (100 nM).

Rotenone stimulates superoxide production by inhibiting complex I of the oxidative phosphorylation system [START_REF] Koopman | Inhibition of complex I of the electron transport chain causes O2-. -mediated mitochondrial outgrowth[END_REF]. The fluorescent oxidation products of non-fluorescent HEt, 2hydroxyethidium and ethidium, were detected in ciPTEC using flow cytometry after harvesting the cells using trypsine/EDTA and washing in PBS. Additionally, one set of samples was pre-incubated with cysteamine (1 mM) for 2.5 hours in culture medium. Data are expressed as mean fluorescence intensity (+/-SEM).

Possible changes in protein oxidation and lipid peroxidation were investigated by determining the amount of butylated protein and F2-isoprostane, respectively. Oxidative stress can lead to the introduction of carbonyl groups in proteins, detectable by immunoblotting using the Oxyblot TM protein oxidation detection kit (Millipore, USA). Matured ciPTEC of 4 controls and 7 patients were examined according to the manufacturer's protocol. After immunoblotting and development using ECL Western blotting substrate (Thermo Fisher Scientific, USA), pixel intensity for each cell line was analysed using ImagePro (MediaCybernetics) and corrected for GAPDH expression.

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Intracellular concentrations of F2-isoprostanes (8-iso-prostaglandin F2α VI; iPF2 α-VI) were determined as marker of oxidative stress using LC-MS/MS. Briefly, butylated hydroxytoluene (0.08% (v/v)) was added to cell pellets to prevent auto-oxidation, and deuterated internal standard (0.2 ng iPF2α-d4, Cayman) was added to correct for loss during sample preparation.

Then, the cells were sonicated for 3x10 sec at 4°C to lyse the cells. Afterwards, KOH was added (final concentration of 1.3 M) and the lysate was incubated for 1 hr at 40°C. Formic acid (20% (v/v)) was added to adjust to pH 4.5 and samples were prepared using solid-phase extraction as reported previously [START_REF] Roest | High levels of urinary F2-isoprostanes predict cardiovascular mortality in postmenopausal women[END_REF]. The eluate was dried under a stream of nitrogen at room temperature and redissolved in 100 µl acetonitrile (10% (w/v)). F2-isoprostanes were analyzed by a 4000 Qtrap (PE Sciex, Canada) MS. The concentration of iPF2α-VI was calculated using a standard curve of iPF2α-VI (Cayman) and corrected for amount of protein.

Data are expressed as ng/mg protein.

ATP metabolism

Intracellular ATP levels were measured in control (21 separate clones of 4 donors) and cystinotic (50 separate clones of 10 donors) matured ciPTEC using ATP Bioluminescence Assay Kit HSII (Roche, Germany) as described before [START_REF] Wilmer | Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells[END_REF]. The effect of cysteamine on intracellular ATP levels was determined in cystinotic ciPTEC (n=4) cultured in presence of cysteamine (1 mM) for 48 hours, with medium replacement every 6 hours.

Mitochondrial and cytosolic ATP production were measured by transfecting proliferating ciPTEC with baculovirus containing cDNA for mitochondria targeted luciferase or luciferase without targeting sequence as described before [START_REF] Valsecchi | Calcium and ATP handling in human NADH:ubiquinone oxidoreductase deficiency[END_REF]. Briefly, proliferating ciPTEC (control n=2, cystinosis n=2; both measured in 4-fold) were cultured on coverslips and transfected for expression of cytosolic or mitochondrial luciferase. After two additional days of culturing at 37°C, ATP production in intact ciPTEC was determined by perfusing the cells with 25 µM luciferin in Hepes-Tris buffer and measuring the luciferase luminescence using

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Phosphate transport and content

Intracellular phosphate levels were determined in duplo in cell pellets of matured control (n=3) and cystinosis (n=6) ciPTEC to test whether decreased ATP levels could be a result of decreased phosphate availability. Data are expressed as mM phosphate / mg protein +/-SEM.

Furthermore, alterations in sodium-dependent phosphate uptake in cystinosis ciPTEC were investigated using radio-labeled 32 PO 4 as describe before [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF]. Km and Vmax values were determined using phosphate saturation curves in control (n=4) and cystinotic (n=5) ciPTEC with GraphPad Prism (version 5.03, GraphPad Sofware Inc.). Further, experiments were performed with a pre-incubation of cysteamine (1 mM) for 2 hours, followed by the uptake of K 2 HPO 4 (0.22 mM) in presence or absence of cysteamine.

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Statistical analysis

To compare cystinosis and control ciPTEC and the effect of cysteamine, appropriate unpaired or paired Student's t-test were used for statistical analysis. Correlations were analyzed using Spearman test.

Results

Cell Culture and proliferation

Primary cell lines derived from urine were successfully transfected using SV40T and hTERT vectors and subcloned as described before [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF]. This resulted in 14 ciPTEC clones, derived from 4 individual control subjects and 10 cystinosis patients (table 1). Activity of alkaline phosphatase activity confirmed proximal tubular origin and was similar in control and cystinosis cell lines (activity in control 2.5 +/-1.6 vs cystinosis 2.3 +/-1.2 mU / mg protein).

Additionally, the expression of the proximal tubular cell markers aminopeptidase N, aquaporin1, P-glycoprotein and dipeptidyl peptidase IV were similar in all cell lines (data not shown). Transfection with SV40T resulted in cell lines proliferating at 33°C, while the disappearance of the SV40T antigen expression at 37°C during 10 days resulted in cell maturation. The additional transfection using hTERT prevented the occurrence of replicative senescence.

Proliferation of control and cystinosis ciPTEC was monitored for 10 days at 37˚C by cell count and protein determination. Both control and cystinosis ciPTEC proliferated similarly up to ~3 days (figure 1a). At this stage, SV40T expression was decreased as previously shown [START_REF] Wilmer | Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters[END_REF]. The total cell population was doubled at the end of the maturation period in both cell populations. Intracellular protein content of both control and cystinosis ciPTEC reached a plateau at day 6 of maturation (figure 1b). Both control and cystinosis ciPTEC readily in protein synthesis (figure 1c). Cell cycle analysis using propidium iodide staining indicated that control and cystinosis cells had similar stages of cell division. At 33°C, 11.0 % (+/-0.5) and 68.3% (+/-2.3) of control ciPTEC compared to 12.8 % (+/-2.5) and 64.3 (+/-5.6) of cystinosis ciPTEC were in the S-phase or G1/G0 phase respectively. After 10 days of maturation at 37°C the percentage of cells in S-phase was decreased to 3.0 % (+/-0.8; p<0.01) and 5.0 % (+/-0.7; p<0.05), respectively. The percentage of G1/G0 phase, indicative for differentiation, increased after maturation to 73.7% (+/-5.9) for control ciPTEC and 70.7%

(+/-5.5) for cystinosis cells. No difference was observed between cystinosis and control cells.

Further, no significant differences in cell division rate nor in intracellular protein content or incorporation were observed between control and cystinosis ciPTEC, allowing the expression of metabolic measurements as a function of protein content in further assays.

Albumin uptake in ciPTEC

Albumin uptake in both control and cystinotic ciPTEC was decreased by megalin-ligand RAP in a concentration dependent manner, suggesting endocytic uptake of albumin via megalin (figure 2a). To test whether albumin uptake was affected in cystinotic ciPTEC, we performed flow cytometry analysis of BSA-FITC uptake in 4 control cell lines and 7 cystinotic cell lines in two independent experiments in triplo (figure 2b). Both experiments demonstrated that albumin uptake in cystinotic cell lines was comparable to control ciPTEC (p=0.75).

Intracellular thiol and disulfide content

Intracellular cystine levels in matured ciPTEC were significantly increased in the cystinosis ciPTEC clones derived from 10 cystinosis patients compared to 4 control ciPTEC (5.2 +/-0.7 versus 0.14 +/-0.02, respectively; p<0.01; figure 3a). Each point represents one patient or control cell line, measured at least in 2 separate assays. The dose-dependent effect of cysteamine on intracellular cystine levels in cystinosis cell lines was determined, demonstrating that 1 mM cysteamine normalized the cystine content within 30 min after

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addition and for a period up to 8 hours (data not shown). By refreshing the cysteamine supplemented culture medium every 6 hours during 48 hours, cystine levels in cystinosis ciPTEC were maintained in the control range (0.26 +/-0.07, p=0.23; figure 3b). Free cysteine levels were also significantly increased in cystinosis ciPTEC (7.2 +/-1.2 versus 2.4 +/-0.5, p<0.05). Similarly, free cysteine levels were normalized upon cysteamine treatment (2.1 +/-0.5, p=0.87; figure 4a).

Total GSH levels were similar in cystinosis and control ciPTEC (34 +/-5 and 35 +/-6, respectively, p=0.86; figure 4b). Remarkably, due to cysteamine treatment for 2 days, total GSH levels were significantly increased in both control (63 +/-10, p<0.05) and cystinosis (95 +/-19; p<0.01) ciPTEC, suggesting that cysteamine treatment increased cellular capacity to deal with oxidative stress (figure 4b).

GSSG levels were significantly increased in cystinosis cells (0.15 +/-0.04 versus 0.66 +/-0.16; p<0.05) and correlated with intracellular cystine levels (p<0.01; r 2 =0.76; figure 4c).

Cysteamine treatment tended to increase GSSG in both control and cystinosis ciPTEC, however this effect was not statistically significant in cystinosis cells. Using the Nernst equation according to Schafer and Buettner [START_REF] Schafer | Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple, Free Radic[END_REF], we estimated the redox status of the GSSG/2GSH couple in ciPTEC. Our calculations revealed that the reduction potential was significantly less negative in matured cystinosis ciPTEC (-270 +/-4 versus -251 +/-1; p<0.01; figure 4d). The reduction potential in cystinosis ciPTEC was restored to control values upon cysteamine treatment (-269 +/-3; p=0.43).

Intracellular ROS production and oxidation status

To get an indication whether increased redox status was a result of increase in ROS production, oxidation was measured under basal and stressed conditions using the probes CM-H 2 DCFDA and HEt . 6a). Although in 5 cystinosis ciPTEC there was a trend towards an increase in intracellular ATP after 48 hours of cysteamine treatment, this increase did not reach statistical significance (p=0.08; figure 6b). To determine whether decreased ATP levels were a result of reduced ATP production, glycolysis and oxidative phosphorylation were Pyruvate production was similar in control and cystinosis cells (figure 6d).

Next, we addressed the possibility that decreased ATP levels could be caused by a decreased phosphate availability. Measurement of intracellular phosphate revealed the absence of any difference between control and cystinosis ciPTEC (0.22 +/-0.04 versus 0.18 +/-0.01, respectively; p=0.18). Additionally, sodium dependent phosphate uptake was determined in ciPTEC, using radio-labeled 32 PO 4 demonstrating decreased phosphate uptake in cystinotic cells (p<0.05) . By analyzing the saturation curves of phosphate uptake in control and cystinotic cells, a Vmax (104.2 +/-15.3 for control and 86.0 +/-5.1 for cystinotic ciPTEC) and Km (0.15 +/-0.08 and 0.20 +/-0.04, respectively) could be determined (figure 7a). The presence of cysteamine (1mM) did not normalize phosphate uptake in cystinotic ciPTEC, despite the normalization of cystine levels (figure 7b).

Discussion and Conclusions

In this study, 4 control and 10 cystinosis conditionally immortalized human cell lines were developed from urine of age matched donors using exfoliated cells of proximal tubular origin.

Using this model, altered GSH redox status and decreased ATP levels were observed.

Moreover, treatment with the cystine depleting agent cysteamine increased intracellular total GSH levels and restored the GSH redox status of the cystinosis cells, but did not normalize affected sodium-dependent phosphate uptake.

The advantage of the presented cell model of cystinosis with different mutations in CTNS

gene is sufficient cystine accumulation, which is ~37-fold higher compared to control cells, which approaches the levels measured in renal tissue [START_REF] Gahl | Cystinosis: a Disorder of Lysosomal Membrane Transport[END_REF]. Furthermore, cystine levels in cystinosis ciPTEC are approximately 6-fold higher compared to a previously reported cystinosis PTEC, immortalized using HPV 16 E6/E7 genes [START_REF] Wilmer | Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells[END_REF]. Probably, the maturation at 37°C, causing decreased cell proliferation, allowed the cells to accumulate more cystine over time. As cells matured for 10 days, continued metabolism and lysosomal protein digestion could result in more cystine trapped in the cystinosis lysosomes. The expression of the temperature sensitive vector SV40T strongly decreased at 37°C allowing cells to differentiate.

As a result, the metabolism of the cells was not (or was less) influenced by the presence of this viral antigen compared to the other cell models.

Additionally, in this study both cystinosis and control cell lines were established using the same methodology from pediatric donors, after similar passage number and in identical phases of the cell cycle, allowing a valuable comparison of their metabolic status. As the study population comprises ciPTEC derived from cystinosis patients with homozygous 57kb deletion and other mutations in the CTNS gene, a genotype-phenotype correlation could be further investigated. However, we neither observed differences in cystine levels nor in ROS production between the two groups of cells. This is coherent with the indistinguishable renal phenotype observed in patients having a homozygous 57kb deletion compared to other truncating mutations.

In the present study, the effect of cysteamine on GSH status was studied for the first time in a renal cystinosis cell model. Remarkably, total levels of GSH increased and the GSH redox status normalized upon cysteamine treatment. In addition to depleting cystine levels, this

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indicates that cysteamine acts as an antioxidant, hence, is a protective agent against oxidative stress. Next to cystine depletion, this mechanism may underlie the protective action of cysteamine against the development of interstitial fibrosis, which leads to end stage renal disease in cystinosis. In line with our findings, Chol et al. [START_REF] Chol | Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines[END_REF] described a trend towards increased levels in GSH upon incubation with cysteamine, ornithine and N-acetyl-cysteine.

As cysteamine restores the GSH redox status, this agent may have beneficial effects in other disorders associated with increased peroxidation, as oxidative damage has been proposed to be involved in the pathogenesis in chronic kidney disease [START_REF] Santangelo | scamps-Latscha, Restoring glutathione as a therapeutic strategy in chronic kidney disease[END_REF][START_REF] Shah | Oxidants in chronic kidney disease[END_REF]. Recently, the use of cysteamine has also been proven to have a synergistic effect on the anti-malarial activity of artemisinin-derivatives, implicating the broader use of this agent [34].

It has been suggested that limited cysteine availability due to dysfunctional cystine efflux from lysosomes in cystinosis was responsible for decreased GSH synthesis via γ-glutamyl cycle [START_REF] Levtchenko | Altered status of glutathione and its metabolites in eystinotic cells[END_REF]. Contrasting this assumption, we and others demonstrated increased intracellular levels of cysteine indicating that limited cysteine availability is not the cause for alterations in γ-glutamyl cycle [START_REF] Bellomo | Modulation of CTNS gene expression by intracellular thiols[END_REF][START_REF] Vitvitsky | The redox status of cystinotic fibroblasts[END_REF]. Elevated free cysteine levels in cystinosis cells can derive from the extracellular medium or synthesis from methionine. A recent study by Bellomo et al. showed that intracellular cysteine levels regulate the CTNS gene expression [START_REF] Bellomo | Modulation of CTNS gene expression by intracellular thiols[END_REF]. Silencing of the CTNS gene was associated with an increase in intracellular cysteine comparable to our results in cystinosis ciPTEC. Interestingly, Foreman et al. demonstrated increased cysteine in CDME loaded rats, which developed renal Fanconi syndrome, without elevated levels of cystine [START_REF] Foreman | Effect of Cystine Dimethylester on Renal Solute Handling and Isolated Renal Tubule Transport in the Rat -A New Model of the Fanconi Syndrome[END_REF].

The mechanism of elevated cysteine in cystinosis proximal tubular cells should be investigated further.

The formation of GSH from lysosomally accumulated cystine upon cysteamine treatment can only partially explain elevated GSH levels, regarding the stochiometry. Free intracellular cysteine (7.2 mole/mg protein) and intracellular cystine (equal to 2 x 5.2 mole/mg protein =

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10.4 mole cysteine/mg protein) in cystinosis ciPTEC are 3.5-fold lower than required for the increase of GSH (95 -34 = 61 mole cysteine/mg protein) upon cysteamine treatment.

Furthermore, proximal tubular GSH transporters might be activated by cysteamine, as tubular GSH levels in vivo are mainly dependent on uptake at the apical and basolateral membrane via specific transporters [START_REF] Lash | Role of glutathione transport processes in kidney function[END_REF][START_REF] Frey | Profiling at mRNA, protein, and metabolite levels reveals alterations in renal amino acid handling and glutathione metabolism in kidney tissue of Pept2-/-mice[END_REF].

In the cystinosis ciPTEC model, the GSH pool is still sufficient to protect proteins and fatty acids from oxidative damage at basal conditions as shown in this study. However, when cystinotic cells are exposed to high levels of oxidation, increased ROS generation is observed.

A similar situation can occur in vivo, because of the active oxidative metabolism in renal proximal tubules, making cystinosis cells prone to the oxidative stress. Further, in vivo increased protein concentration in the tubular lumen can exaggerate oxidative stress leading to progressive tubulo-interstitial inflammation [START_REF] Zandi-Nejad | Why is proteinuria an ominous biomarker of progressive kidney disease?[END_REF]. In this situation, intracellular GSH availability can be of significant importance and, therefore, optimal cysteamine treatment protecting against peroxidation can improve renal survival. In addition, due to the prominent role of the mitochondrial ATP production, an increased ROS production compared to the in vitro model can be expected requiring more GSH for protection. Hence, alterations in GSH metabolism may induce apoptotic cell death and cause mitochondrial oxidative damage [START_REF] Franco | SLCO/OATP-like transport of glutathione in FasLinduced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis[END_REF][START_REF] Park | Lysosomal cystine storage augments apoptosis in cultured human fibroblasts and renal tubular epithelial cells[END_REF]. Therefore, the various hypotheses with involvement of GSH metabolism, ATP metabolism or enhanced cell death may represent different facets of a unique cascade leading to tubular dysfunction initiated by lysosomal cystine accumulation [START_REF] Park | Increased apoptosis in cystinotic fibroblasts and renal proximal tubule epithelial cells results from cysteinylation of protein kinase Cdelta[END_REF][START_REF] Sansanwal | Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis[END_REF].

In contrast to our results, a recent study in fibroblasts showed intact redox status as presented by GSSG/2GSH ratio, with normal total GSH levels [START_REF] Vitvitsky | The redox status of cystinotic fibroblasts[END_REF]. The latter study correctly pointed towards the difficulties in measuring disulfides and mentioned a possible underestimation of the total GSH pool in the fibroblasts previously measured by our group [START_REF] Levtchenko | Altered status of glutathione and its metabolites in eystinotic cells[END_REF]. Taken into account that 1 mg protein corresponds to a cell volume of 5 µl [START_REF] L'allemain | Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport[END_REF], the levels measured in GSH levels measured in cultured ciPTEC in the current study are comparable to the levels measured in tubular cells in vivo, which are reported to be in the range of 5 mM [START_REF] Lash | Role of glutathione transport processes in kidney function[END_REF].

Furthermore, in the current study, the cysteine pool is about 10-fold lower than the GSH pool in control ciPTEC, which is comparable to other studies [START_REF] Vitvitsky | The redox status of cystinotic fibroblasts[END_REF][START_REF] Bellomo | Modulation of CTNS gene expression by intracellular thiols[END_REF]. The observed variability in the intracellular thiol concentration between individual cell lines points to the necessity to analyze sufficient number of cell lines for making statistically valuable comparisons.

In our study, decreased levels of intracellular ATP in cystinosis ciPTEC were observed. Both glycolytic and mitochondrial ATP production were intact, suggesting that decreased ATP levels were a result of increased ATP consumption. A possible mechanism by which ATP consumption can be increased in cystinosis was recently suggested by Kumar et al. [START_REF] Kumar | A futile cycle, formed between two ATP-dependant gamma-glutamyl cycle enzymes, gamma-glutamyl cysteine synthetase and 5oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?[END_REF]. They proposed that a futile turnover of 5-oxoproline to glutamate in the absence (or decreased availability) of cysteine, consuming two additional ATP molecules per cycle, might be responsible for the decreased ATP and explain elevated excretion of 5-oxoproline into cystinosis urine. As we found no decreased levels of intracellular cysteine in cystinosis, this mechanism, in our opinion, is not explaining the observed decrease in ATP. Alternatively, a defect in the Na-dependent 5-oxoproline transporter in renal proximal tubules might be responsible for the increased excretion of this metabolite [START_REF] Miyauchi | Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter[END_REF] The artificial loading with CDME was recently used by Figueiredo et al. to demonstrate decreased adenylate cyclase activity [START_REF] Figueiredo | Cysteamine prevents inhibition of adenylate kinase caused by cystine in rat brain cortex[END_REF]. They suggested that cystine inhibition of this enzyme, catalyzing the phosphate transfer between ATP and AMP, could be partially responsible for the observed ATP decrease in cystinosis cells. In our present study, we showed that cysteamine treatment did not improve cellular phosphate uptake despite slight increase of intracellular ATP levels, implicating that elevated cystine levels are not directly associated defective proximal tubular reabsorption. The latter observation corresponds to persistence of Fanconi syndrome in vivo

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 21
in cystinosis patients despite cysteamine therapy [START_REF] Wilmer | Cystinosis: practical tools for diagnosis and treatment[END_REF]. Moreover, these findings demonstrate again that the CDME loading technique is of limited value in studying the pathogenesis of cystinosis [START_REF] Wilmer | Cystine Dimethylester Model of Cystinosis: Still Reliable?[END_REF].

Defective albumin uptake, another feature of Fanconi syndrome, could not be demonstrated in the cystinotic ciPTEC model. This is in line with our previous study demonstrating both intact apical expression of megalin together with abundant endocytic vesicles containing albumin and other megalin ligands in human cystinotic kidney tissue [START_REF] Wilmer | Urinary Protein Excretion Pattern and Renal Expression of Megalin and Cubilin in Nephropathic Cystinosis[END_REF]. Thus, the origin of proteinuria can be different in cystinosis compared to other pathological conditions manifesting with renal Fanconi syndrome, such as Dent disease, in which apical expression of megalin in PTEC is decreased [START_REF] Christensen | Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules[END_REF].

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

  Abbreviations:

incorporated 3 H

 3 -Leu between day 8-10 of maturation, indicating the absence of any difference A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 13

A

  C C E P T E D M A N U S C R I P T investigated in intact matured cells expressing either cytosolic or mitochondria targeted luciferase. Neither cytosolic (glycolysis) nor mitochondrial (oxidative phosphorylation) ATP production was altered in cystinosis cells (p values of 0.18 and 0.60, respectively; figure 6c). Using glycolysis inhibitors DOG and SIA, ATP levels were decreased (SIA: >60%; DOG >90%) in one control and two cystinosis cell lines, indicating ciPTEC derive their ATP mainly from glycolysis. Since cultured ciPTEC are mainly glycolytic, we further explored the glycolytic activity. Both inhibitors significantly decreased the production of pyruvate in control (DOG: p<0.05; SIA: p<0.05) and cystinosis (DOG: p<0.05; SIA: p<0.01) ciPTEC.

  are 35 nmol GSH / mg protein / 5µl and equals to 7 mM. Hence,

Figure 1 .

 1 Figure 1. Cell proliferation of control and cystinotic ciPTEC. (A) Cell proliferation

Figure 2 .

 2 Figure 2. Albumin uptake in ciPTEC. (A) Albumin uptake in both control and cystinotic

Figure 3 .Figure 4 .

 34 Figure 3. Cystine accumulation in ciPTEC. (A) HPLC analysis reveals significantly

Figure 5 .

 5 Figure 5. Intracellular ROS production in ciPTEC. (A) Basic CM-H 2 DCFDA oxidation is

Figure 6 .Figure 7 . 31 Fig. 1 A 32 Fig. 2 A 33 Fig. 3 A 34 Fig. 4

 67311322333344 Figure 6. ATP metabolism in ciPTEC. (A) Intracellular levels of ATP are significantly

Fig. 5 A 36 Fig. 6 A

 5366 Fig. 5

Fig. 7 A

 7 Fig. 7

  3 H-Leucine ( 3 H-Leu) incorporation. Control and cystinotic ciPTEC were cultured in triplo in 24 well plates in the presence of 3 H-Leu (3 µCi/ml; GE Healthcare, UK) supplemented tissue culture medium for

	2, 6, 16 and 24 hrs. Cells were washed using ice-cold PBS and proteins were precipitated
	using 10% (w/v) trichloroacetic acid. Proteins were dissolved in 400 µl NaOH (0.3 M) and
	subsequently to neutralization with 80µl HCl (1.5M) radioactivity was measured using a
	TriCarb liquid scintillation counter (Perkin Elmer, USA). Using specific activity of 3 H-Leu,
	incorporation of Leu was calculated and expressed as pmol/mg protein. Cell cycle analysis
	was performed on proliferating and matured cells (control n=3; cystinosis n=6), by harvesting
	cells using trypsin and fixation in 70 % (v/v) ice-cold ethanol. Subsequently, cells were
	stained using propidium iodide (10 µg/ml) in citrate buffer (0.2 M Na 2 HPO 4 , 0.1 M citric
	acid) in the presence of RNAse (5 µg/ml). Cell cycle was determined by flow cytometry and
	presented as percentage of the cells in the S-phase of mitosis.

  incubated in the absence and presence of H 2 O 2 (0; 10; 100 and 1000 µM) as a positive control to stimulate oxidation and to verify the sensitivity of the assay. Formation of CM-DCF as a marker for oxidation was monitored immediately and monitored for 15 min on Victor3V multilabel counter (ex464 nm, em530 nm; Wallac, Perkin Elmer). Additionally, one set of samples was pre-incubated with 1 mM cysteamine for 2.5 hrs at 37°C. Basal and H 2 O 2 stimulated CM-H 2 DCF oxidationwas calculated as the slope of fluorescence intensity increase versus time and expressed as oxidation rate (+/-SEM) with untreated cells set at 100% after correction for protein content.

	ACCEPTED MANUSCRIPT
	M A N U S C R I P T
	A C C E P T E D
	. Matured ciPTEC were washed in HEPES-Tris buffer (NaCl (132 mM), KCl (4.2 mM),
	CaCl 2 (1 mM), MgCl 2 (1 mM), D-glucose (5.5 mM), Hepes (10 mM), pH7.4 using Tris) and
	incubated for 15 min in the dark with 80 µl CM-H 2 DCFDA (10 µM). Esterase hydrolyzes
	CM-H 2 DCFDA to form intracellular trapped CM-H 2 DCF, which can oxidize to fluorescent
	CM-DCF, e.g. by the action of ROS. Cells were washed twice in HEPES-Tris buffer and
	9

  R I P T photomultiplier tube. After reaching a stable signal, cells were perfused with digitonin (DIG; 100 µM) and excess ATP (10 mM). Permeabilization of cellular membranes by DIG in the presence of excess ATP resulted in a peak in luminescence signal, which was limited by total luciferase present in the cells. Basal cytosolic and mitochondrial ATP production are expressed as percentage of the peak in luminescence after DIG treatment (+/-SEM).

	Cytosolic ATP production from glycolysis was measured by determining pyruvate production
	in conditioned culture medium of ciPTEC and by measuring ATP levels in the presence of
	inhibitors of glycolysis [8,31]. Briefly, matured ciPTEC (control n=5; cystinosis n=8) were

cultured in serum-free culture medium for 2.5 hrs in presence or absence of glycolysis inhibitors sodium iodoacetate (SIA; 0.02 mM) or deoxy-glucose (DOG; 100 mM). Pyruvate production was subsequently measured in culture medium using lactate dehydrogenase (100 U/ml; Roche, Germany) after addition of NAD + (2 mM; Roche, Germany) at pH 9.5. Formation of NADH is directly correlated to glycolytic pyruvate production and was analyzed by measuring the absorbance at 340 nm on Victor3V Multilabel Counter (Wallac, Perking Elmer, USA).

  Henk Blom of VU Amsterdam and Sjoerd Verkaart of the Radboud University Nijmegen Medical Centre and Francesco Emma of the Bambino Gesù Children's Hospital Rome, Italy, for fruitful discussions. Measurement of intracellular phosphate levels under supervision of Prof. Hans Willems is appreciated. Technical assistance by Dinny van Oppenraaij-Emmerzaal, Jenneke Keizer-Garritsen and Gwendolyn Beckmann is gratefully acknowledged.

Taken together, the identical preparation of four control and ten cystinosis ciPTEC in the present study allows valid and detailed in vitro investigation of cell metabolism involved in the pathogenesis of cystinosis. Using this model, increased oxidation of GSH was demonstrated resulting in altered intracellular redox status in cystinosis cells. Cysteamine increased intracellular GSH levels and restored redox status of GSSG/2GSH couple. This mechanism may be responsible for better conservation of renal function in cystinosis patients treated with cysteamine. The increase of intracellular GSH in control cells upon cysteamine treatment points to the possibility of using this amino thiol in other renal disorders associated with enhanced oxidative stress.

Table 1 . Clinical information and CTNS mutation of the patients from whom ciPTEC

 1

	were derived. ND, not detected in the coding region or promotor of CTNS gene; m, male; f,
	female. 2 [53]

Acknowledgements

The work presented in this study was performed thanks to financial support from Cystinosis Research Foundation, Cystinosis Research Network,funding from European Community´s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 201590 and Fund for Scientific Research, Flanders (F.W.O. Vlaanderen) (grant 1801110N). The authors thank


Jos C F M Dortmans

Guus Koch

Peter J M Rottier

Ben P H Peeters
email: ben.peeters@wur.nl

A comparative infection study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion

Keywords: Newcastle disease virus (NDV), pigeon paramyxovirus type I (PPMV-1), pigeons, pathogenesis

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical symptoms were only observed in the FL-Herts infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection.

However, only the AV324/96 and rgAV324 infected birds, and not the FL-Herts infected animals, shed virus in the cloaca. The AV324/96 infected pigeons showed higher mean antibody titres than the rgAV324 infected animals, whereas the antibody titres of the FL-Herts infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.

Introduction

Newcastle disease (ND) is a highly contagious viral disease of chickens but can also affect a wide variety of other avian species including domestic pigeons and doves (Columbiformes) [START_REF] Kaleta | Newcastle disease in free-living and pet birds[END_REF]. The disease is caused by Newcastle disease virus (NDV) or avian paramyxovirus type 1 (APMV-1), which belongs to the family Paramyxoviridae (genus Avulavirus) [START_REF] Mayo | A summary of taxonomic changes recently approved by ICTV[END_REF]. During the 1980s, a disease caused by an APMV-1 spread worldwide among racing and show pigeons (Alexander et al., 1985). APMV-1 variants like this could be discriminated antigenically by monoclonal antibodies [START_REF] Russell | Antigenic variation of Newcastle disease virus strains detected by monoclonal antibodies[END_REF]Alexander et al., 1984;Alexander et al., 1985;[START_REF] Lana | Characterization of a battery of monoclonal antibodies for differentiation of Newcastle disease virus and pigeon paramyxovirus-1 strains[END_REF]Collins et al., 1989) and genetically by phylogenetic analysis of the F gene nucleotide sequence [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF] and have become known as pigeon paramyxovirus type 1 (PPMV-1).

In the past, a number of ND outbreaks in poultry have been attributed to PPMV-1, which makes these pigeon-derived viruses a real and continuous threat to the poultry industry [START_REF] Anonymous | Newcastle disease: outbreaks linked to pigeons[END_REF]Alexander et al., 1985;[START_REF] Alexander | Newcastle disease outbreak in pheasants in Great Britain in May 1996[END_REF][START_REF] Alexander | Newcastle disease outbreaks in domestic fowl and turkeys in Great Britain during 1997[END_REF][START_REF] Werner | Characterization of avian paramyxovirus type 1 strains isolated in Germany during 1992 to 1996[END_REF][START_REF] Liu | Characterization of pigeon-origin Newcastle disease virus isolated in China[END_REF]Irvine et al., 2009). The F proteins of PPMV-1 strains contain a poly-basic cleavage site motif, a feature generally associated with virulent NDV strains. It has therefore been suggested that PPMV-1 strains have evolved from virulent NDV strains [START_REF] Meulemans | Antigenic and biological characterization of avian paramyxovirus type 1 isolates from pigeons[END_REF]Collins et al., 1996;[START_REF] Ujvari | Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission[END_REF]. However, some PPMV-1 strains cause only minimal disease and show a low intracerebral pathogenicity index (ICPI) in chickens [START_REF] Meulemans | Evolution of pigeon Newcastle disease virus strains[END_REF]. Nevertheless, they are potentially virulent as becomes apparent after serial passages in chickens [START_REF] Alexander | Avian paramyxovirus type 1 infections of racing pigeons: 2 pathogenicity experiments in pigeons and chickens[END_REF]Collins et al., 1994;[START_REF] Kommers | Virulence of pigeonorigin Newcastle disease virus isolates for domestic chickens[END_REF][START_REF] Kommers | Virulence of six heterogeneous-origin Newcastle disease virus isolates before and after sequential passages in domestic chickens[END_REF]Dortmans et al., 2010a). Thus, In our recent studies, we investigated the molecular characteristics of PPMV-1 isolate AV324/96, originally isolated from a racing-pigeon. Despite having an F protein cleavage site motif typical of virulent viruses, this isolate is low virulent for chickens (ICPI=0.44, as determined by the Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom). Using a reverse-genetics system we investigated the contribution of the F, NP, P, M and L proteins to virulence for chickens by exchanging genes between this AV324/96 strain and the highly virulent NDV Herts strain (Dortmans et al., 2009;Dortmans et al., 2010b). Furthermore, we investigated the molecular basis for the virulence increase of AV324/96 after serial passage in chickens (Dortmans et al., 2010a). In view of the demonstrated potential of infected pigeons transmitting NDV to poultry, more knowledge about these PPMV-1 viruses is needed quite urgently. One important aspect is the pathogenesis of PPMV-1 in its original host, the pigeon. Therefore we investigated the susceptibility of pigeons to the PPMV-1 isolate AV324/96 and to a recombinant derivative prepared from it (rgAV324). For comparison we also used the recombinant APMV-1 strain FL-Herts which is highly virulent for chickens.

Materials and Methods

Viruses. Experimental design. Three groups of 5 pigeons each were inoculated with virus by the combined intraocular, intranasal and intratracheal route, using a total dose of 10 7

EID 50 /pigeon. The negative control group consisted of 4 pigeons which received PBS.

The experiment was terminated at 14 days post infection (dpi).

The animals were observed for clinical signs, and tracheal and cloacal swabs were taken daily. The swabs were placed in 2.95% tryptose phosphate buffer with appropriate antibiotics and infectious virus was quantified by serial end-point dilution in 96-well plates using QM5 cells [START_REF] Antin | Isolation and characterization of an avian myogenic cell line[END_REF]. After 3 days of incubation at 37ºC, cells were fixed and immunological staining was performed to detect the infected cells as previously described (Dortmans et al., 2009). Virus titres were calculated using the Reed and Muench method [START_REF] Reed | A simple method of estimating fifty percent endpoints[END_REF] and expressed as log 10 TCID 50 /ml.

Serum samples were obtained at day 6 before and day 14 after inoculation. The hemagglutination inhibition (HI) assay was performed by standard methods [START_REF] Alexander | Newcastle Disease[END_REF] using 8 hemagglutinating units of antigen. The antigens used in this study were rgAV324, FL-Herts and strain Ulster. The latter is used as an antigen in standard NDV diagnostics.

Results

Clinical signs. all pigeons had positive tracheal swabs from 1 to 5 dpi. Pigeon 7738 incurred a dislocated beak at 3 dpi; hence, a throat swab had to be taken instead of a tracheal swab, which may explain the negative results at 3 and 5 dpi for this particular animal.

Two pigeons in the rgAV324 infected group (7737 and 7738) and one in the group infected with AV324/96 (7746) were positive until 9-10 dpi. No differences in the amounts of virus shedding were observed among the three virus groups (Fig. 1). This was confirmed by estimating and summing up the area under the curve of each individual animal per group (p > 0.05; data not shown). Surprisingly, although all pigeons in the FL-Herts infected group became virus positive in the trachea, none of these birds showed detectable virus in the cloacal swabs. In contrast, three pigeons from both the rgAV324 and the AV324/96 infected group became virus positive in the cloaca (Table 1 and Fig. 1). Thus, although the PPMV-1 virus did not cause any clinical symptoms, infected pigeons shed virus until at least 14 dpi.

Serology.

To evaluate the antibody responses of the animals to the infections, serum samples were obtained at day 6 before and day 14 after inoculation. Serum samples taken before inoculation were used to confirm the lack of NDV-specific HI antibodies of the pigeons. At 14 dpi, using the homologous antigens, all pigeons of the rgAV324 and AV324/96 infected group responded positively in the HI test, whereas one pigeon of the FL-Herts infected group did not respond (Table 2). AV324/96 infected pigeons showed a broader range of reactivity compared to the animals in the rgAV324 infected group. Notably, FL-Herts infected pigeons did not show any cross reactivity with the rgAV324 antigen.

Discussion

From the results of this study we conclude that pigeon strain AV324 is not virulent for pigeons, while FL-Herts is only modestly virulent for pigeons. Although we did not infect chickens via the natural mucosal route, the ICPI values suggest that the difference in virulence of APMV-1 and PPMV-1 is much smaller in pigeons than in chickens. This indicates that chickens and pigeons are not equally susceptible to infection by the same strain of virus, and confirms the findings of others (Erickson et al., 1980;[START_REF] Kapczynski | Susceptibility and protection of naive and vaccinated racing pigeons (Columbia against exotic Newcastle disease virus from the California 2002-2003 outbreak[END_REF][START_REF] Wakamatsu | Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003[END_REF][START_REF] Carrasco | Experimental infection of Newcastle disease virus in pigeons (Columba livia): humoral antibody response, contact transmission and viral genome shedding[END_REF].

Several earlier studies have shown that pigeons infected with either APMV-1 or PPMV-1 shed virus via the oral, tracheal or laryngeal route from 2 dpi onwards (Duchatel & Vindevogel, 1986;Duchatel et al., 1992;[START_REF] Vindevogel | Paramyxovirus type 1 infection in pigeons[END_REF][START_REF] Kapczynski | Susceptibility and protection of naive and vaccinated racing pigeons (Columbia against exotic Newcastle disease virus from the California 2002-2003 outbreak[END_REF][START_REF] Wakamatsu | Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003[END_REF]. Cloacal shedding was also observed in most of these studies, but the onset of this shedding was generally much earlier, starting at 2-3 dpi (Duchatel & Vindevogel, 1986;[START_REF] Stone | Efficacy of oil-emulsion vaccines prepared with pigeon paramyxovirus-1, Ulster, and La Sota Newcastle disease viruses[END_REF]Duchatel et al., 1992;[START_REF] Vindevogel | Paramyxovirus type 1 infection in pigeons[END_REF][START_REF] Kapczynski | Susceptibility and protection of naive and vaccinated racing pigeons (Columbia against exotic Newcastle disease virus from the California 2002-2003 outbreak[END_REF][START_REF] Wakamatsu | Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003[END_REF]Carrasco et al., 2009), except in one case where cloacal virus shedding was observed from 5-11 dpi [START_REF] Carrasco | Experimental infection of Newcastle disease virus in pigeons (Columba livia): humoral antibody response, contact transmission and viral genome shedding[END_REF], comparable to our results (6-11 dpi).

While, in these studies, virus shedding was determined either by inoculating embryonated eggs or chicken embryo fibroblast cultures or by RT-PCR, we used QM5 cells for our infection assay, which may be less sensitive. Alternatively, the AV324/96 strain might replicate at a lower rate than the other investigated viruses, hence resulting in a later onset of viral shedding via the cloacal route.

The lower antibody response to rgAV324 infection compared to infection with its parental virus AV324/96 might be explained by the broader quasispecies distribution of the latter. This difference in quasispecies complexity might also explain the difference in ICPI value we observed between recombinant and parental viruses in chickens. These results also show that using standard diagnostics with Ulster antigen, low antibody responses against PPMV-1 in pigeons could be missed.

Differences in HI antibody titres depending on the virus strain used as the antigen in the HI assay have been noted before [START_REF] Stone | Efficacy of oil-emulsion vaccines prepared with pigeon paramyxovirus-1, Ulster, and La Sota Newcastle disease viruses[END_REF][START_REF] Vindevogel | Paramyxovirus type 1 infection in pigeons[END_REF][START_REF] Maas | The use of homologous virus in the haemagglutination-inhibition assay after vaccination with Newcastle disease virus strain La Sota or Clone30 leads to an over estimation of protective serum antibody titres[END_REF][START_REF] Kapczynski | Susceptibility and protection of naive and vaccinated racing pigeons (Columbia against exotic Newcastle disease virus from the California 2002-2003 outbreak[END_REF]Hu et al., 2009). Such differences might well relate to observations suggesting that current ND vaccines may provide less protection due to antigenic divergence between the vaccine strain and the circulating field strains [START_REF] Kapczynski | Protection of chickens against overt clinical disease and determination of viral shedding following vaccination with commercially available Newcastle disease virus vaccines upon challenge with highly virulent virus from the California 2002 exotic Newcastle disease outbreak[END_REF][START_REF] Qin | Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996 to 2005[END_REF][START_REF] Van Boven | Herd immunity to Newcastle disease virus in poultry by vaccination[END_REF]Hu et al., 2009;[START_REF] Miller | Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses[END_REF]. PPMV-1 strains belong to a distinct genetic lineage [START_REF] Aldous | A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons[END_REF] and previous work already suggested that vaccines for use in pigeons should preferably be prepared from pigeon-origin PPMV-1 virus [START_REF] Alexander | Avian paramyxovirus type 1 infections of racing pigeons: 4 laboratory assessment of vaccination[END_REF].

To assess the virulence of the different viruses used in this study, the ICPIs were determined in day-old chickens. The ICPI value for AV324/96 was 0.2, which is lower than the value for the original isolate which was 0.44. This difference might be explained by the fact that the ICPI assays were performed at different institutes using slightly different virus preparations. Although the ICPI is a standardized method, minor variations such as the genetic background of the birds used, difference in reading clinical signs and or small differences in the exact time of onset of clinical signs may result in small differences in the ICPI value for viruses of low virulence.

Furthermore, the virus inocula used at both locations might have differed slightly with respect to the composition of subpopulations. Although PPMV-1 or velogenic NDV infections in pigeons can cause morbidity and mortality, we found strain AV324 not to be virulent at all. Yet, despite the lack of clinical symptoms, the animals do excrete virus at levels that are comparable to those obtained with virulent viruses for both chickens and pigeons [START_REF] Vindevogel | Paramyxovirus type 1 infection in pigeons[END_REF][START_REF] Kapczynski | Susceptibility and protection of naive and vaccinated racing pigeons (Columbia against exotic Newcastle disease virus from the California 2002-2003 outbreak[END_REF][START_REF] Wakamatsu | Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003[END_REF][START_REF] Carrasco | Experimental infection of Newcastle disease virus in pigeons (Columba livia): humoral antibody response, contact transmission and viral genome shedding[END_REF]Carrasco et al., 2009). Although mortality of chickens infected with velogenic NDV can reach 100%, mortality of pigeons is usually less than 40% and for PPMV-1 it is even less than 10% [START_REF] Marlier | Viral infections in pigeons[END_REF]. However, some experimental and field data show higher mortality rates (Erickson et al., 1980;Duchatel & Vindevogel, 1986). A reason for this might be that the pigeons are suffering from secondary bacterial or viral infections [START_REF] Marlier | Viral infections in pigeons[END_REF]. In pigeon experiments, SPF animals are rarely used, in contrast to chicken experiments.

PPMV-1 is capable of being transmitted from infected pigeons to chickens placed in contact [START_REF] Alexander | Avian paramyxovirus type 1 infections of racing pigeons: 2 pathogenicity experiments in pigeons and chickens[END_REF] and requires only a limited number of adaptive mutations for optimal replication in chickens (Dortmans et al., 2010a) Tropical Animal Health and Production, 41, 1325-1333. Collins, M.S., Alexander, D.J., Brockman, S., Kemp, P.A. & Manvell, R.J. (1989).

Evaluation of mouse monoclonal antibodies raised against an isolate of the variant avian paramyxovirus type 1 responsible for the current panzootic in pigeons.

Archives of Virology, 104, 53-61. Collins, M.S., Strong, I. & Alexander, D.J. (1996)

  study of pigeon and avian paramyxovirus type 1 viruses in pigeons: evaluation of clinical signs, virus shedding and seroconversion

  from pigeons to chickens, PPMV-1 strains may evolve into virulent viruses and lead to major outbreaks.

Fig. 1 .Fig. 1 .

 11 Fig. 1. Virus titres determined in tracheal (left column) and cloacal (right column)

  Carrasco, A.O., Seki, M.C., de Sousa, R.L., Raso, T.F. & Pinto, A.A. (2009). Protection levels of vaccinated pigeons (Columba livia) against a highly pathogenic Newcastle disease virus strain.

	animal technicians for performing the animal experiments. We also thank Dr. M.S.
	Collins (Veterinary laboratories Agency, Addlestone, Surrey, UK) for providing
	pigeon isolate AV324/96. This research was financed by the Dutch Ministry of
	Agriculture, Nature and Food Quality.
	F o r F o r F o r
	P P P
	e e e
	e r e r e r
	R R R
	e e e
	. This knowledge, together with the recent NDV outbreaks in pigeons caused by PPMV-1 in v i e v i e v i e Europe (www.oie.int/wahis/public.php?page=disease_immediate_summary), w w w
	underscores the potential danger of pigeon-derived paramyxoviruses for the poultry industry. O n l O n l O n l
	y y y

  . Pathogenicity and phylogenetic evaluation of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses" based on the nucleotide sequence of the fusion protein gene. Archives of Virology, 141, 635-647. Collins, M.S., Strong, I. & Alexander, D.J. (1994). Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses". Archives of Virology, 134, 403-411. Communities, C.o.t.E. (1992). Council directive 92/66/EEC of 14 July 1992 introducing community measures for the control of Newcastle disease. Official Journal of the European Communities, L260, 1-20. de Leeuw, O.S., Koch, G., Hartog, L., Ravenshorst, N. & Peeters, B.P. (2005). Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein. Journal of General Virology, 86, 1759-1769. Dortmans, J.C., Rottier, P.J., Koch, G. & Peeters, B.P. (2010a). Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence. Journal of General Virology, vir.0.026344-0. Avian Pathology, 21, 321-325. Duchatel, J.P. & Vindevogel, H. (1986). Assessment of vaccination of pigeons against paramyxovirus type 1 infection with inactivated aqueous-suspension or oilemulsion vaccines. Avian Pathology, 15, 455-465. Erickson, G.A., Brugh, M. & Beard, C.W. (1980). Viscertropic velogenic Newcastle disease in pigeons: clinical disease and immunization. Avian Diseases, 24, 257-267. Hu, S., Ma, H., Wu, Y., Liu, W., Wang, X., Liu, Y. & Liu, X. (2009). A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics. Vaccine, 27, 904-910. Irvine, R.M., Aldous, E.W., Manvell, R.J., Cox, W.J., Ceeraz, V., Fuller, C.M., Wood, A.M., Milne, J.C., Wilson, M., Hepple, R.G., Hurst, A., Sharpe, C.E., Alexander, D.J. & Brown, I.H. (2009). Outbreak of Newcastle disease due to pigeon

	Dortmans, J.C., Rottier, P.J., Koch, G. & Peeters, B.P. (2010b). The viral replication complex is associated with virulence of Newcastle disease virus. Journal of Virology, 84:10113-10120. Dortmans, J.C., Koch, G., Rottier, P.J. & Peeters, B.P. (2009). Virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein. Journal of General Virology, 90, 2746-2750. Duchatel, J.P., Flore, P.H., Hermann, W. & Vindevogel, H. (1992). Efficacy of an inactivated aqueous-suspension Newcastle disease virus vaccine against paramyxovirus type 1 infection in young pigeons with varying amounts of o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w maternal antibody. F O n l O n l O n l O n l O n l
	y y y y y

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

Acknowledgements

We would like to thank Lisette Cornelissen for discussions about the experimental set up, Kees Westerink and Heleen Klos for breeding and providing the pigeons and the


Emma Teirlynck

Maarten De Gussem

Jeroen Dewulf

Freddy Haesebrouck

Richard Ducatelle

Filip Van Immerseel
email: filip.vanimmerseel@ugent.be

Vetworks Dewulf

M De Gussem²

R Dycatelle

Morphometric evaluation of 'dysbacteriosis' in broilers

Keywords: dysbacteriosis, broilers, morphometric evaluation, intestinal health

come   L'archive ouverte pluridisciplinaire

Introduction

Since the ban of antimicrobial growth promoters (AGPs) in Europe, the broiler industry is facing a rise in intestinal health problems, collectively referred to as 'dysbacteriosis' among practitioners [START_REF] Eshuis | Bacterie-regelaar Tylan helpt tegen dysbacteriose[END_REF][START_REF] Fabri | Necrotic enteritis, Clostridial enteritis or dysbacteriosis[END_REF]Rebel et al., 2006;[START_REF] De Gussem | Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health[END_REF].

Dysbacteriosis has been defined as the presence of a qualitatively and/or quantitatively abnormal microbiota in the proximal parts of the small intestine, inducing a cascade of reactions in the gastro-intestinal tract including reduced nutrient digestibility and impaired intestinal barrier function, increasing the risk of bacterial translocation and inflammatory responses [START_REF] Fabri | Necrotic enteritis, Clostridial enteritis or dysbacteriosis[END_REF]Panneman, 2000;van der Klis & Lensing, 2007).

The syndrome is generally seen between 20 and 30 days of age [START_REF] Fabri | Necrotic enteritis, Clostridial enteritis or dysbacteriosis[END_REF]Pattison, 2002;Wilson et al., 2005). Clinically, the main symptoms are: pale, glistening or orange droppings with undigested feed particles, wet and greasy droppings and hence dirty feathers, sometimes foamy cecal droppings, reduced physical activity, increased water intake, a decrease in feed intake with a check in weight or reduced gain rates and an increased feed conversion [START_REF] Fabri | Necrotic enteritis, Clostridial enteritis or dysbacteriosis[END_REF]Pattison, 2002;Wilson et al., 2005;[START_REF] De Gussem | Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health[END_REF]. At necropsy the main observations are thin, fragile, often translucent intestinal walls, watery or foamy intestinal contents and frequent orange mucus and undigested particles in the intestines, ballooning of the gut and intestinal inflammation (Pattison, 2002;[START_REF] De Gussem | Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health[END_REF].

It is believed that both non-infectious and infectious factors can play a role in dysbacteriosis (Mortimer, 2002;[START_REF] De Gussem | Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health[END_REF]. Suspected non-infectious factors are different types of non-specific stressors, such as feed interruptions or dietary changes, nutritional imbalance, dietary stressors such as soluble 'non-starch polysaccharides' (NSP), [START_REF] Langhout | Effect of dietary high-and low-methylated citrus pectin on the activity of the ileal microflora and morphology of the small intestinal wall of broiler chicks[END_REF][START_REF] De Gussem | Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health[END_REF]Teirlynck et al., 2009). Infectious agents that potentially play a role in dysbacteriosis are coccidia, Clostridium perfringens and other unidentified bacteria producing toxic metabolites (Morrow, 2001;[START_REF] De Gussem | Coccidiosis in poultry: review on diagnosis, control, prevention and interaction with overall gut health[END_REF].

Despite the widespread nature and importance of the syndrome, there is a lack of scientific literature and still a lot of controversy about the exact etiology and up until today the basis of the underlying pathophysiology is unknown. Hence, diagnosis faces a lot of challenges in broilers due to the incomplete characterization of the syndrome (Wilson et al., 2005).

The purpose of the present study was to gain insight in the histopathological changes at the level of the intestinal mucosa in field cases of dysbacteriosis.

Materials and methods

Animals and sampling. Twelve 18 days old (2 flocks, 1 broiler farm), thirty-eight 21 days old (4 flocks, 2 broiler farms), thirty-six 28 days old (2 flocks, 1 broiler farm) and eighteen 42 days old (1 flock, 1 broiler farm) male Ross 308 broiler chickens were used in the study.

In total broilers of nine flocks from four different broiler farms were sampled. The flocks have been selected ad random from an integrated poultry enterprise, thus all the birds originated from the same breeder flocks and were given similar feed including anticoccidials.

The birds were receiving nicarbazin/narasin in starter phase (day 1 until day 18) and salinomycin in grower phase until 30 days of age at registered doses. This enterprise was reported by the field veterinarian to have above average intestinal health problems, including management was evaluated as better than average. The birds that were selected were not clinically ill, and were considered as average, healthy birds by an experienced poultry veterinarian.

The chickens were euthanized by means of cervical dislocation by an experienced veterinarian and immediately necropsied. Intestinal parameters were scored (see below).

Samples of approximately 3 cm were taken from the second limb of the duodenum and the middle part of one caecum, rinsed in PBS and fixed in 4% (v:v) buffered formalin. 1) Each bird was given a score between 0 and 10 for intestinal dysbacteriosis parameters, 0 being a normal gastrointestinal tract and 10 being the most severe dysbacteriosis. In total 10 parameters were assessed and scored 0 when absent and 1 when present. The parameters are: (1) ballooning of the gut; (2) significant redness of the serosal and/or mucosal side of the gut and/or presence of abnormally dilated blood vessels on the serosal side of the gut, cranial from Meckel's diverticulum; (3) a macroscopic visible and/or tangible reduction of the gut wall thickness and/or translucent guts in combination with increased fragility of the gut cranial from Meckel's diverticulum;

Macroscopical scoring system. (Figure

(4) when 3 seconds after dissecting the gut, the edges of the gut cranial from Meckel's diverticulum are flaccid; (5) an abnormal appearance of the contents in the lumen of the gut (excessive slime, water, gas, greasy aspect or mixture of these) cranial from Meckel's diverticulum; (6) significant redness of the serosal and/or mucosal side of the gut and/or presence of abnormally dilated blood vessels on the serosal side of the gut, caudal from Meckel's diverticulum; (7) a macroscopic visible and/or tangible reduction of the gut wall thickness and/or translucent guts in combination with increased fragility of the gut caudal from Meckel's diverticulum; (8) when 3 seconds after dissecting the gut, the edges of the gut caudal from Meckel's diverticulum are flaccid; (9) abnormal appearance of the contents in the lumen of the gut (excessive slime, water, gas, greasy aspect or mixture of these) caudal from Meckel's diverticulum; (10) undigested feed particles caudal from ileo-caecal junction [START_REF] De Gussem | Macroscopic scoring system for bacterial enteritis in broiler chickens and turkeys[END_REF]. During this lesion scoring, no intestinal sections were given score 0, 9 or 10.

Morphological examination. Formalin fixed intestinal segments were dehydrated in xylene, and embedded in paraffin. Sections of 4 µm were cut using a microtome (Microm, Prosan, Merelbeke, Belgium). Deparaffinization was done in xylene (2 x 5 min). Then the sections were rehydrated in isopropylene (5 min), 95% alcohol (5 min) and 50% alcohol (5 min) and stained with haematoxylin and eosin. Histological lesions were studied using standard light microscopy. Villus length in duodenum was measured by random measurement of 9 villi per section using an Olympus BX61 Digital Camera DP50 (Olympus NV, Aartselaar, Belgium) and a computer based image analysis system, Analysis® J-2 (P4 technologies, inc., Waldorf, Maryland, US). Only intact villi were measured, meaning villi for which the tip as well as the base of the villus were in the plane of the section. Thickness of the tunica muscularis in duodenum and ceca was also measured using the Analysis® J-2 software. For each section 8 measurements were performed on different locations. Measurements were done on cross sections of ring shaped intestinal segments which allow unbiased perpendicular measurements. Detection of goblet cells was done using Periodic Acid Schiff staining as described by [START_REF] Forder | Bacterial modulation of small intestinal goblet cells and mucin composition during early posthatch development of poultry[END_REF] Immunohistochemical examination. Deparaffinization of paraffin-embedded tissue sections (4 µm) was performed as described above. The pressure cooker antigen retrieval method (Tender Cooker, Nordic Ware, Minneapolis, USA) was applied to the samples. Immunohistochemical labelling of leukocytes was performed as described by [START_REF] Mast | Characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01[END_REF]. Briefly, endogenous peroxidase in the tissue sections was blocked with H 2 O 2 (3%) in methanol for 30 min (21°C). After rinsing with PBS sections were incubated for 1 h (21°C) with monoclonal antibodies directed against T-lymphocytes (KUL05). After rinsing thoroughly, a goat anti-mouse IgG1 conjugate, labeled with peroxidase (Dako, Heverlee, Belgium) was added for 30 min (21°C). After rinsing again, tissue sections were incubated with ABC HRP complex (Dako, Heverlee, Belgium) for 30 min (21°C). After another rinse, positive cells were stained brown after conversion of the substrate (3,3' diaminobenzidine tetrahydrochloride, Sigma, St. Louis, USA) in the presence of H 2 O 2 . The number of Tlymphocytes in the propria mucosae was scored with an automatic image analysis system (Optimas 6.5., Media Cybernetics, Silver Spring, USA), measuring the area percentage occupied by the labeled cells. For each section, eight randomly selected sites were analyzed by the image analysis program. account. First, all independent variables were tested univariably and subsequently all significant variables were included in a multivariable model. In this multivariable model also all 2-way interactions were tested. In the multivariable model all main effects and interactions with a p-value <0.05 were retained. For the outcome variables that were not normally distributed, a log transformation was performed. Model fit was evaluated by means of evaluation of the residuals. The number of chickens belonging to a certain macroscopical dysbacteriosis score group per age group is shown in Table 1.

Statistical analysis. The relation between the macroscopic lesion

Results

Intestinal morphology. Villus length. Duodenal villus length generally decreased with an increase in the macroscopic 'dysbacteriosis' score within one age group (Table 2). Statistical analysis showed that, starting from macroscopical dysbacteriosis score 5, villus length was significantly lower compared to macroscopical dysbacteriosis score 1 (p=0.01). Score 8 differed significantly from score 5, 6 and 7 (p=0.03).

Cystic crypts.

In the duodenum a mild to severe dilatation of the crypts of Lieberkühn was observed. Although not quantitatively analyzed the number of cystic crypts appeared to increase with age and with the macroscopic dysbacteriosis score. The age effect seemed to have the highest impact.

Thickness of the tunica muscularis. For each age interval, the thickness of the tunica muscularis generally decreased with an increasing macroscopical dysbacteriosis score (Table 3). Statistical analysis showed that the thickness of the tunica muscularis of the gut of the animals having macroscopical dysbacteriosis score 5 and higher was significantly lower as compared to score 1 (p=0.01).

Goblet cells.

Although not quantitatively analyzed, periodic acid schiff staining generally showed more and larger goblet cells both on the villi and in the crypts of the gut of animals with higher macroscopical dysbacteriosis scores.

T-lymphocyte infiltration. Generally T-lymphocyte infiltration in duodenum and caecum increased within one age-group, with increased macroscopical dysbacteriosis scores (Table 4). Statistical analysis showed that T-lymphocyte infiltration in the gut mucosa of animals having a macroscopical dysbacteriosis score of 6 and higher was significantly higher as compared to score 1 (p=0.02).

Discussion

Definitive diagnosis of dysbacteriosis has been challenging due to the non-specific nature of the clinical signs and lesions and because the etiology is still unknown (Wilson et al., 2005).

The present study has identified several more or less characteristic changes, which may aid in confirming the diagnosis of 'dysbacteriosis', and which help differentiating this entity from other intestinal disorders, such as malabsorption syndrome (MAS) and runting-stunting syndrome (RSS).

MAS has been described as a gastro-intestinal disease affecting broilers during the et al., 2002;van Hemert et al., 2004). The main clinical symptoms of MAS are weight gain depression with non-uniform growth, stunting, diarrhea with undigested food particles resulting in wet litter, retarded and defective feathering, pigment loss, distended abdomens, depression and early mortality. At necropsy, lesions are found in the digestive organs, especially the small intestine, which is grossly pale and distended, with mucoid contents, and there is lower mineralization of the thigh bones (Kouwenhoven et al., 1978a, b;[START_REF] Bracewell | The infectious stunting syndrome[END_REF]Reece et al., 1984;Szabo et al., 1989;Reece & Frazier, 1990;McNulty & McFerran, 1993;Sályi & Glávitis, 1999;Songserm et al., 2002a, b;Van Hemert et al., 2004;Rebel et al., 2006). Histologically, intestinal lesions such as cystic crypts of Lieberkühn and villus atrophy are observed (Rebel et al., 2004).

RSS is typically affecting birds within the first two weeks of life and can be defined as a syndrome in which a number of individuals in a flock appear considerably small due to delayed growth [START_REF] Dufour-Zavala | Cystic enteritis: reproduction of the disease and attempted control measures[END_REF]Nili et al., 2007). Clinically, the main symptoms of RSS are immobility, increased feed conversion and poor performance, uneven growth and stunting, little eating and excessive water consumption, diarrhea, retarded and defective feathering, distended abdomens and bone defects (Vertommen et al., 1980a, b;Ruff, 1982;[START_REF] Calnek | Diseases of Poultry 10th edn[END_REF]Shapiro et al., 1997;1998;Sályi and Glávits, 1999;[START_REF] Dufour-Zavala | Cystic enteritis: reproduction of the disease and attempted control measures[END_REF]Nili et al., 2007). At necropsy lesions are found in the small intestine, which is pale and thin, almost translucent, containing undigested food particles.

A main difference between dysbacteriosis and MAS/RSS, of which the latter could be identical entities with different names, is that in field cases of dysbacteriosis, the growth retardation due to poorer absorption of nutrients is probable but very often average growth formation could be due to a vitamin deficiency as a secondary effect of the malabsorption caused by villus atrophy and increased T-lymphocyte infiltration [START_REF] Klasing | Nutritional Diseases[END_REF].

Dysbacteriosis is defined in human medicine as a condition characterized by a shift in the microbiota favoring abnormal populations of bacteria to predominate within the intestinal tract with minimal intestinal pathology (Schoorel et al., 1980;[START_REF] Dumitrasco | Dysbacteriosis in enteropathies[END_REF]Sidorchuk & Bondarenko, 1984;[START_REF] Klemparskaya | Normalizing effect of immunoglobulins in the treatment of endogenous infection and intestinal dysbacteriosis in irradiated mice[END_REF]. Despite the definition of dysbacteriosis, the microbiota composition and possible microbiota shifts have not been studied and the name dysbacteriosis is not really substantiated. However there are indications suggesting that the intestinal microbiota composition may play a role in this syndrome.

These indications include the response to certain antibiotic treatments, the consistency of the droppings and the inflammatory nature of the intestinal lesions.

In conclusion, dysbacteriosis in broilers is characterized by villus atrophy, a decrease in the thickness of the tunica muscularis and an increase in T-lymphocyte infiltration in the gut mucosa. These histological observations can explain performance problems and macroscopical observations of the gut at necropsy, in broilers with dysbacteriosis. Vertommen, M., Vaneck, J.H.H., Kouwenhoven, B. & Vankol, N. (1980a). Infectious stunting and leg weakness in broilers. Pathology and biochemical-changes in blood-plasma.

Avian Pathology, 9, 133-142. Vertommen, M., Vanderlaan, A. & Veenendaalhesselman, H.M. (1980b). Infectious stunting and leg weakness in broilers. Studies on alkaline-phosphatase iso-enzymes in blood-plasma.

  genetic background, enzymatic dysfunction, and mycotoxins

  (mainly Eimeria maxima) and wet litter, although the (ventilation)

  score and the outcome variables villus length, thickness of the tunica muscularis and T-lymphocyte infiltration was evaluated by means of a linear mixed model taking into account the chicken as a random factor since multiple observations were made on one animal. In addition other co-variables such as age of the bird, person scoring the bird and intestinal segment were also taken into

  post-hatch (Zekarias

  in line with breed standards, and also the homogeneity of the flock will typically not be dramatically affected as with RSS/MAS. Also the age of disease induction is lower (below 2 weeks of age) in MAS/RSS as compared to dysbacteriosis (3-4 weeks of age).Dysbacteriosis and MAS/RSS are clearly distinct from subclinical necrotic enteritis because no ulcerations in the gut are found.In the dysbacteriosis field cases, duodenal villus length, and thickness of the tunica muscularis and T-lymphocyte infiltration in the mucosa of duodenum and caecum were altered. No necrotic lesions were observed. This combination of morphological and inflammatory changes combined with the different clinical appearance compared to other syndromes (such as MAS/RSS) is in our opinion sufficiently unique and characteristic to classify this syndrome as a separate entity, although microscopical measurements of MAS/RSS samples are necessary for confirmation. The microscopical findings of dysbacteriosis may explain the clinical signs, being depressed growth, wet litter and clinical depression(Toskes et al., 1975;[START_REF] Kaldhusdal | Barley inclusion and Avoparcin supplementation in broiler diets. 2. Clinical, pathological, and bacteriological findings in a mild form of necrotic enteritis[END_REF][START_REF] Hoerr | Intestinal integrity and the impact of losing it[END_REF]. Based on the measurements, variations in intestinal distention between healthy gut and severe dysbacteriosis can explain the variations in thickness of the tunica muscularis. Thus ballooning of the gut and the flaccid aspect of the gut wall may be directly associated with reduced tonus / rigor of the tunica muscularis. Variations in intestinal distention however can only partly explain the reduction in villus length and increase in villus thickness as observed in the severe dysbacteriosis cases. Thus severe dysbacteriosis is associated with an absolute reduction in available absorptive surface area. The change in the size and number of goblet cells could explain the mucous content of the intestinal tract of affected birds. Cystic crypt

  .S. & McFerran, J.B. (1993). The runting stunting syndrome general assessment. In J.B. McFerran & M.S. McNulty (Eds.). Virus infections of birds (pp. 519-528). Elsevier, Amsterdam. Morrow, C. (2001). Solving the problems of necrotic enteritis. British Poultry Science, 42, 64-68. Mortimer, I. (2002). The Detection of Dysbacteriosis. Proceedings of the Elanco Global Enteritis Symposium. Cambridge, UK. Nili, H., Jahantigh, M. & Nazifi, S. (2007). Clinical observation, pathology, and serum biochemical changes in infectious stunting syndrome of broiler chickens. Comparative Clinical Pathology, 16, 161-167. Panneman, H. (2000). Clostridial enteritis/dysbacteriosis, fast diagnosis by T-RFLP, a novel diagnostic tool, Proceedings of the Elanco Global Enteritis Symposium. Cork, Ireland. Pattison, M. (2002). Some clinical and pathological features of enteritis in broilersobservations on treatment in the UK. Proceedings of the Elanco Global Enteritis Symposium (pp.1-7). Cambridge, UK. Rebel, J.M.J., van Dam, J.T.P., Zekarias, B., Balk, F.R.M., Post, J., Minambres, A.F. & ter Huurne, A.A.H.M. (2004). Vitamin and trace mineral content in feed of breeders and their progeny: effects of growth, feed conversion and severity of malabsorption syndrome of broilers. British Poultry Science, 45, 201-209.Rebel, J.M.J., Balk, F.R.M., Post, J.,Van Hemert, S., Zekarias, B. & Stockhofe, N. (2006).Malabsorption syndrome in broilers. World's Poultry Science Journal, 62,[17][18][19][20][21][22][23][24][25][26][27][28][29]

  .L., Hooper, P.T., Tate, S.H., Beddome, V.D., Forsyth, W.M.,Scott, P.C. & Barr, D.A. (1984). Field, clinical and pathological observations of a runting and stunting syndrome in broilers. VeterinaryRecord, 115, 483-485. Reece, R.L. & Frazier, J.A. (1990). Infectious stunting syndrome of chickens in Great-Britain -field and experimental studies. AvianPathology, 19, 723-758. Ruff, M.D. (1982). Nutrient Absorption and Changes in Blood-Plasma of Stunted Broilers.AvianDiseases, 26, 252-259. Sályi, G. & Glávits, R. (1999). Infectious stunting syndrome associated with disturbances of mineral metabolism and bone development in broiler chickens. Acta Veterinaria Hungarica, 47, 361-378.Schoorel, E.P., Giesberts, M.A.,Blom, W. & Van Gelderen, H.H. (1980). D-Lactic acidosis in a boy with short bowel syndrome. Archives of Disease in Childhood, 55, 810-812. Shapiro, F., Mahagna, M. & Nir, I. (1997). Stunting syndrome in broilers: Effect of glucose or maltose supplementation on digestive organs, intestinal disaccharidases, and some blood metabolites. Poultry Science, 76, 369-380. Shapiro, F., Nir, I. & Heller, D. (1998). Stunting syndrome in broilers: Effect of stunting syndrome inoculum obtained from stunting syndrome affected broilers, on broilers, leghorns and turkey poults. Poultry Science, 77, 230-236. Sidorchuk, I.I. & Bondarenko, V.M. (1984). Selection of a biologically active mutant of Propionibacterium shermanii and the possibility of its use in complex therapy of enteral dysbacteriosis. Journal of Hygiene, Epidemiology, Microbiology and Immunology, 28, ., Engel, B., van Roozelaar, D.J., Kok, G.L., Pijpers, A., Pol, J.M.A. & ter Huurne, A.A.H.M. (2002a). Cellular immune response in the small intestine of two broiler chicken lines orally inoculated with malabsorption syndrome homogenates. Veterinary Immunology and Immunopathology, 85, 51-62. Songserm, T., Zekarias, B., van Roozelaar, D.J., Kok, R.S., Pol, J.M.A., Pijpers, A. & ter Huurne, A.A.H.M. (2002b). Experimental reproduction of malabsorption syndrome with different combinations of reovirus, Escherichia coli, and treated homogenates obtained from broilers. Avian Diseases, 46, 87-94. Szabo, J., Salyi, G. & Rudas, P. (1989). Effect of malabsorption-syndrome on pancreatic function in broilers. Poultry Science, 68, 1553-1560. Teirlynck, E., Bjerrum, L., Eeckhaut, V., Huygebaert, G., Pasmans, F., Haesebrouck, F., Dewulf, J., Ducatelle, R. & Van Immerseel F. (2009). The cereal type in feed influences gut wall morphology and intestinal immune cell infiltration in broiler chickens. British Journal of Nutrition, 102, 1453-1461. Toskes, P.P., Giannella, R.A., Jervis, H.R., Rout, W.R. & Takeuchi, A. (1975). Small intestinal mucosal injury in the experimental blind loop syndrome; light and electronmicroscopic and histochemical studies. Gastroenterology, 68, 1193-1203. van der Klis, J.D. & Lensing, M. (2007). Wet litter problems relate to host-microbiota interactions. World Poultry, 23, 20-22.Van Hemert, S., Hoekman, A.J., Smits, M.A. & Rebel, J.M.J.(2004). Differences in intestinal gene expression profiles in broiler lines varying in susceptibility to malabsorption syndrome. PoultryScience, 83, 1675Science, 83, -1682. .

Table 1 .

 1 Avian Pathology, 9, 143-152. Wilson, J., Tice, G., Brash, M.L. & St Hilaire, S. (2005). Manifestations of Clostridium perfringens and related bacterial enteritides in broiler chickens. Worlds Poultry Science Journal, 61, 435-449. Zekarias, B., Songserm, T., Post, J., Kok, G.L., Pol, J.M.A., Engel, B. & ter Huurne, A.A.H.M. (2002). Development of organs and intestinal mucosa leukocytes in four broiler lines that differ in susceptibility to malabsorption syndrome. Poultry Science, 81, 1283-1288. Number of chickens analyzed per age per macroscopic dysbacteriosis group. Of each chicken both duodenum and caecum were analyzed.-: No birds of this age group were given this score.

	Age	Macroscopic 'Dysbacteriosis' Score
		1		2	3	4	5	6	7	8
	18 days 21 days	F o -r -	1 3	4 4	4 13	3 4	-8	-5	-1
	28 days	P 1	3	-	3	13	6	6	4
	42 days	1	r e e 1 1	2	4	6	1	2
					R e v i e w
							O n
								l y
										19

Table 2 .

 2 Length of villi (µm) in duodenal sections in relation to the macroscopic score. No birds of this age group were given this score.

	Random measurements of 9 villi per 3 gut sections were performed by a computer based
	analysis system. The given values ± standard deviations are the means of 9 villi measured in
	the chickens from the same age and in the same macroscopic 'dysbacteriosis' score
	category.
	Age	Macroscopic 'Dysbacteriosis' Score
		1	2	3	4	5	6	7	8
		-	1988	1668	1627	1550	-	-	-
	18 days		± 153	± 219	± 188	± 201
		-	2125	1929	2006	1870	1694	1491	1495
	21 days		± 260	± 239	± 292	± 219	± 54	± 193	± 126
		2656	1866	-	2173	1878	1812	1762	1479
	28 days	± 245	± 152		± 269	± 290	± 415	± 336	± 212
		2439	2471	2847	2364	2049	2078	2527	1862
	42 days	± 151	± 81	± 222	± 185	± 208	± 289	± 190	± 180

-:

Table 3 .

 3 Thickness of the tunica muscularis (µm) in duodenal and caecal sections in relation to the macroscopic score. 8 random measurements of the thickness of the tunica muscularis per 3 gut sections were performed by a computer based analysis system. The given values ± standard deviations are the means of 8 random measurements of both duodenum and caecum of the chickens in the same age and in the same macroscopic 'dysbacteriosis' score category. -: No birds of this age group were given this score.

	Age	Macroscopic 'Dysbacteriosis' Score
		1	2	3	4	5	6	7	8
		-	197	152	143	119	-	-	-
	18 days		± 22	± 28	± 25	± 16
		-	233	186	178	174	144	144	127
	21 days		± 40	± 30	± 39	± 33	± 16	± 27	± 23
		293	306	-	253	211	185	170	151
	28 days	± 76	± 105		± 62	± 52	± 37	± 26	± 23
		327	329	277	246	205	193	195	165
	42 days	± 61	± 91	± 45	± 77	± 29	± 45	± 21	± 30

Table 4 .

 4 T-lymphocyte infiltration (area percentage) for duodenal and caecal sections in relation to the macroscopic score. 8 random measurements of the T-lymphocyte infiltration in the mucosa per 3 gut sections were performed by a computer based analysis system. The given values ± standard deviations are the means of the 8 random measurements of both duodenum and caecum of the chickens in the same age and in the same macroscopic 'dysbacteriosis' score category. -: No birds of this age group were given this score Content of the intestinal tract, 1. Mucoid, orange intestinal content, 2. Foamy intestinal content; C. Tonus of the intestinal tract, 1. Good tonus, 2. Lack of tonus; D. Macroscopically visible thickness of the intestinal tract, 1. Macroscopically thin intestinal tract, 2. Intestinal tract with normal thickness; E. Undigested particles in the colon (arrows); F. Inflammation of the gut, 1. Inflammation, 2. No inflammation.

	Age	Macroscopic 'Dysbacteriosis' Score
		1	2	3	4	5	6	7	8
		-	4.72	5.64	5.54	6.52	-	-	-
	18 days		± 1.70	± 2.36	± 2.14	± 3.31
		-	4.06	5.05	5.56	5.10	6.30	6.80	8.20
	21 days		± 1.70	± 2.01	± 2.64	± 2.35	± 3.10	± 3.05	± 1.95
		5.59	7.93	-	7.70	7.48	10.24	8.74	11.00
	28 days	± 1.80	± 3.88		± 3.19	± 2.83	± 3.19	± 3.66	± 3.06
		7.82	6.97	8.44	5.57	7.24	8.61	7.69	9.50
	42 days	± 2.35	± 1.47	± 2.20	± 1.72	± 2.58	± 2.71	± 1.33	± 2.01

23 Figure 1. Macroscopic dysbacteriosis score system parameters. A. Overall gut ballooning; B.

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

Acknowledgments

We would like to thank Sarah Loomans, Christian Puttevils and Delphine Ameye for their skilful technical assistance. Dieter Vancraeynest and Maja Mariën (Alpharma) are acknowledged for their help with macroscopical lesion scoring. This work was funded by grant S6169 of the Federal Public Service Health, Food Chain Safety and Environment.