Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis. Leishmania cause a spectrum of cutaneous manifestations ranging from limited cutaneous lesions that heal spontaneously to the more severe mucocutaneous form. These different clinical manifestations depend on the species of Leishmania and the host immune response [Leishmania major. This mouse strain develops progressive lesions, uncontrolled growth of parasites, visceralization, and death. The C57BL/6 strain is resistant to infection by L. major, controls parasite replication, and heals lesions [L. major is mediated by IFN-γ, TNF-α, and activation of macrophages to produce nitric oxide [Parasites of the genus response , 2. In e lesions . Howeveric oxide –6.L. braziliensis. It is characterized by control of parasite growth in the tissue, but persistent chronic inflammation that commonly affects mucosal tissue causing severe disfiguration and social stigma to the patient [γ and TNF-α, are found in these patients [L. braziliensis causes a very mild and self-limited lesion in C57BL/6 and BALB/c strain mice [L. amazonensis causes a persistent chronic lesion in C57BL/10 and C57BL/6 mice that lasts over 20 weeks, but both animal models fail to control the parasite in the tissue, a hallmark of mucocutaneous leishmaniasis. Moreover, IFN-γ and TNF production is impaired in these infection models [L. major. TNFR1 KO mice control tissue parasitism similarly to the wild-type resistant mouse, but develop nonhealing lesions. However, these lesions do not increase in size progressively. On the contrary, they remain chronic and small, but last for at least 20 weeks afterinfection [Mucocutanesous leishmaniasis is caused mainly by  patient . High copatients . The stuain mice , 10. In n models –13. The nfection , 15. L. major, TNF-α is important for activation of macrophages, in cooperation with IFN-γ, and elimination of intracellular parasites [L. major-infected mice [α may play a key role in the healing of L. major lesions. However, this important phenomenon was not described in the chronic stage of infection to explain the persistent lesions in L. major-infected TNFR1 KO. Thus, the aim of this study was to characterize events in the chronic phase of L. major infection in TNFR1 KO mice. In experimental infection by arasites , 16–18. ted mice . This in C57BL/6 wild-type (WT) mice, 6 to 10 weeks old, were obtained from CEBIO . TNFR1 KO mice were originally obtained from the University of Pennsylvania  and maintained in Laboratory of the Gnotobiology and Immunology of the Instituto de Ciências Biológicas . All the procedures involving animals were in accordance with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation and were approved by the UFMG animal experimentation ethical committee at UFMG (CETEA), protocol number 55/2009.Leishmania major (WHO MHOM/IL/80/Friedlin) was used in this study. Parasites were maintained in Grace's insect medium , pH 6.2, supplemented with 20% fetal bovine serum , 2 mM l-glutamine , 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO BRL Life Technologies). Mice were injected in the hind footpads with 1 × 106 L. major metacyclic promastigotes. Footpads were measured weekly with a caliper . Lesion sizes are expressed as the difference between infected and uninfected footpads.A clone of Parasite load in infected footpads was determined by limiting dilution . Resultsμm thick sections were stained with hematoxylin and eosin and analyzed by light microscopy. At least 10 microscopic fields measuring 250,000 micrometers, representative of lesions, were automatically analysed by KS 300  for determining the comparative cellularity of lesions at 15 wks.Infected footpads from WT and TNFR1 KO mice were removed at 6 and 15 weeks after infection and fixed in 10% of formalin. Tissues were processed and embedded in paraffin and 5 in situ by the TUNEL reaction, an in situ cell death detection kit . The results were obtained by counting the number of stained cells per 100 cells  in 6–10 random areas per histological section.Infected footpads from WT and TNFR1 KO mice were removed at 6 and 11 weeks after infection and fixed in 4% formaldehyde. Apoptotic cells were assessed α (53-6.7), CD3 (17A2), F4/80 (BM8), and Ly6G (RB6-8C5) in PBS containing 1% FBS for 20 min on ice and then washed and fixed with 2% formaldehyde. Stained cells were analyzed using an FACScan flow cytometer equipped with cellQuest software . Statistical analyses of mean fluorescence intensity (MFI) were performed using the FlowJo v7.6.5 software .Infected footpads from WT and TNFR1 KO mice were removed at 6 and 15 weeks after infection. Collected tissues were incubated for 90 minutes with 1.5 mg/mL of collagenase  in RPMI 1640 without supplements at 37°C, homogenized using a tissue grinder and centrifuged at 2000 g. Single-cell suspensions were stained with fluorochrome-conjugated antibodies  against CD4 (RM4-5), CD8 L. major-infected and noninfected footpads were harvested and placed in 0.5 mL TRIZOL (GIBCO BRL) solution and homogenized, and RNA extraction was performed according to the manufacture's instruction. RNA was quantitated spectrophotometricaly and 1 μg of RNA was reverse transcribed using 25 U of M-MLV Reverse Transcriptase  in 12.5 μL reaction containing 2.5 mM dNTPs, 50 mM TRIS/HCl (pH8.3), 75 mM KCl, 2 mM MgCl2, 10 mM DTT , 250 mM dNTP (Promega), 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 10 U RNAsin (GIBCO BRL), and 7.5 pM of oligo dT15 (GIBCO BRL). The mixtures were incubated for 5 min at 95°C, 5 min at 4°C, and 5 min at 25°C; at this step 25 U of reverse transcriptase was added to each sample and the reaction mixture was incubated for 60 min at 37°C. The temperature was then elevated to 95°C for 5 min and cooled again at 4°C for 5 min. The PCR was performed in 10 μL reaction of samples diluted in the following buffer: 250 mM dNTP, 10 mM Tris-HCl pH 8.3, 50 mM KCL, 1.5 mM MgCl2, 5 mM of each primer and 0.05 U of TaqPolimerase . For each pair of primers, at least three different numbers of cycles were tested and the best nonsaturating condition was chosen. The PCR was standardized using the house-keeping gene HPRT. The primer sequences (sense and antisense sequence), PCR product size, number of cycles, and temperature of annealing used for each primer are listed in 2HPO4, pH 7.2) containing antiproteases  and 0.05% tween-20. Homogenized tissues were kept on ice for 30 min and subsequently centrifuged (3000 × g for 10 min). The preparations were assayed using RD Systems kits  according to instructions. The sensitivity for CCL-2 and CCL-5 was 16 and 32 pg/mL, respectively.Chemokines and cytokines were analyzed by two methods: detection of mRNA by reverse transcription polymerase chain reaction (RT-PCR) and ELISA, at different times of infection . The footpad was excised and total protein and RNA were extracted with Trizol (GIBCO BRL Laboratories), as previously described. Cytokine and chemokine detections by RT-PCR were performed as previously described . BrieflyL. major-infected TNFR1 KO mice and attempted to explain the reason why these lesions fail to heal. As shown previously [L. major-infected TNFR1 KO mice develop chronic nonhealing lesions, while WT mice heal lesions over time , which is induced by IFN-γ and TNF-α [γ and TNF-α present in lesions from TNFR1 KO mice infected with L. major, it is not surprising that these mice control parasite growth. The reason for discrepancy between mRNA and protein expression of TNF-α is not known, but we could speculate that there is posttrancriptional regulation or that protein is accumulated at the site of infection, since there is no signaling through TNFR1. Albeit TNF-α was shown to signal through TNFR1 to induce NOS2, alternative pathways in TNFR1 KO mice have been proposed [α [Resolution of nd TNF-α –6. Givenproposed , 29, 30.proposed , and it posed [α , 15, we α is the starting factor that mediates cell infiltration to a site of infection or tissue damage [L. braziliensis who developed mucocutaneous leishmaniasis [TNF-e damage . Accordie damage . In othee damage , 32–35. e damage –38. Likemaniasis , 26. We L. major in TNFR1 KO is related with high concentrations of these two chemokines that are promoting the recruitment of more cells. Cell recruitment to sites of infection or tissue damage is mediated by chemokines . In an aRhodococcus equis. Moreover, cells from R. equis-infected lungs of WT mice and from footpads of L. major-infected WT were sensitive only to TNF-mediated apoptosis. However, cells from TNFR1 KO mice infected with either R. equis or L. major were not sensitive to TNF-α-mediated apoptosis [in situ, in lesions from WT and TNFR1 KO mice. At 6 weeks of infection, both mouse strains showed similar concentrations of TUNEL positive cells, thus suggesting that at this time point apoptosis occurred independently of the TNFR1. However, at 11 weeks of infection, when WT mice were healing and TNFR1 KO mice had a large inflammatory infiltrate at the site of infection, lesions from WT mice had significantly more TUNEL positive cells than lesions from TNFR1 KO mice. This indicated to us that TNFR1 plays a crucial role in the clearance of the inflammatory infiltrate and in the healing of lesions. Apoptosis is required for the clearance of inflammatory cells from tissues, once the stimulus for recruitment of pathogens or damage is gone , 45. TNFpoptosis . Thus, wL. major as a model to study chronic nonhealing lesions found in patients that present the mucocutaneous clinical form of leishmaniasis (In this paper, we implicated persistent chemokine production and defective apoptosis as the factors that prevent healing of lesions in TNFR1 KO mice. In addition, characterization of parasite burden, cellular infiltrate, and cytokine production allowed us to propose the TNFR1 KO infected with maniasis .


Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG).A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets.P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall  and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO−.MPO+ and CD15+ cell infiltration were significantly correlated . However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC. Outgrowth and progression of human colorectal cancers (CRC) are driven by gene mutations and microsatellite instability tumor inherent characteristics Infiltration by activated CD8+ memory T cells and expression of IFN-γ gene within CRC were convincingly shown to be associated with favorable prognosis The role of innate immune system cells was not studied in comparable detail and controversial data were reported regarding CRC infiltration by NK cells Granulocytes have largely been disregarded by tumor immunologists In previous work, we showed that CRC infiltration by CD33+/HLA-DR−/CD16+ myeloid cells is associated with improved patient survival Myeloperoxidase (MPO) is a lysosomal enzyme produced in high amounts by neutrophilic granulocytes (NG) In a small series of CRC samples (n = 67), it has been shown that MPO+ cell infiltration is significantly higher in CRC than in normal colon mucosa CD15, also known as Lewis X and stage-specific embryonic antigen 1, is a carbohydrate adhesion molecule expressed on mature neutrophils, mediating phagocytosis and chemotaxis Here we show for the first time that a subgroup of CRC is characterized by a high infiltration by MPO+ and CD15+ positive cells. Most importantly, high MPO+ cell density in CRC is independently associated with favorable prognosis.Written consent has been given from the patients for their information to be stored in the hospital database and used for research. The use of this clinically annotated TMA for research was approved by the corresponding Ethics Committee of the University Hospital of Basel (Ethikkommission beider Basel) and the ex vivo analyses were approved by the Institutional Review Board (63/07). For freshly excised clinical specimens included in this study written consent has been given from the patients undergoing surgical treatment at Basel University Hospital.The TMA used in this work was constructed by using 1420 non-consecutive, primary CRCs, as previously described Clinicopathological data for the 1420 CRC patients included in the TMA were collected retrospectively in a non-stratified and non-matched manner. Annotation included patient age, tumor diameter, location, pT/pN stage, grade, histologic subtype, vascular invasion, border configuration, presence of peritumoral lymphocytic inflammation at the invasive tumor front and disease-specific survival . Tumor bOverall survival was defined as primary endpoint. Follow-up data were available for 1379 patients with mean/median and IQR event-free follow-up time of 67.7/68 and 45–97 months.Standard indirect immunoperoxidase procedures were used for immunohistochemistry . Briefly, slides were dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum  for 20 min and incubated with primary antibody at room temperature. Primary antibodies used were specific for MPO , CD15 , CD16 , CD68 , FOXP3  and CD8 . Subsequently, sections were incubated with peroxidase-labelled secondary antibody (DakoCytomation) for 30 min at room temperature. For visualization of the antigen, sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen (DakoCytomation) for 30 min, and counterstained with Gill’s hematoxylin.MPO+ and CD15+ tumor infiltrating cells were counted for each punch (approximately one high power [20x] field) by a trained research fellow [R.D.]. Data were independently validated by two additional investigators [L.To. and C.H.] and a high Spearman correlation coefficient ( = 0.82) and a highly significant (p<0.0001) correlation between measurements was observed. Evaluation of MLH1, MSH2, MSH6, CD16, CD68, CD8 and FOXP3 specific stainings in the CRC TMA under investigation was published previously Following Institutional Review Board approval (63/07), tissues from surgically removed CRC and adjacent normal mucosa were minced, centrifuged, and resuspended in RPMI 1640 medium supplemented with 5% foetal calf serum, 2 mg/ml collagenase IV, 0.1 mg/ml hyaluronidase V, and 0.2 mg/ml DNAse I . Following a 1 hour digestion, cell suspensions were filtered and centrifuged. For phenotypic analysis of surface markers, cells were stained with mAbs for 15 minutes on ice in PBS, washed once with PBS 0.5% FCS, 0.5 M EDTA buffer and fixed in lysis buffer from BD Bioscience (1∶10). Samples were then permeabilized in BD fixation/permeabilization buffer. For intracellular staining, an anti-MPO reagent or an isotype matched negative control antibody were added for a 15 min incubation at room temperature. After a PBS wash, cells were suspended in wash buffer and analyzed by flow cytometry using a 2-laser BD FACSCalibur . Results were analyzed by Cell Quest  and Flow Jo  softwares.Cut-off scores used to classify CRC with low or high MPO+ or CD15+ infiltration were obtained by regression tree analysis, evaluating sensitivity and false positive rate for the discrimination of survivors and non-survivors, on all tumor samples http://www.R-project.org). Data reporting was performed according to the REMARK criteria Spearman’s rank correlation was used to analyze the correlation between MPO+, CD15+, CD16+, CD68+, CD8+ and FOXP3+ cell infiltration. Statistical analyses were performed using R i386 Version 2.15.2  infiltration by MPO+ and CD15+ cells was detectable in CRC samples  and mean: 16.4, median: 7, range 0–125 cells/punch for CD15 (n = 1191), respectively; Normal colonic mucosa was indeed infiltrated by MPO+ and CD15+ cells  and mean: 4.3, median: 1, range 0–35 cells/punch for CD15 (n = 47), respectively). However, a significantly higher  were used to assess clinicopathological correlations.P = 0.04). Most importantly, it was significantly associated with early (pT1–2) tumor stage (P = 0.007), absence of local recurrence (P = 0.031) and higher 5-year survival rate (P = 0.0009) , detectable in 14.5% of tumors, was significantly associated with older age of patients ( 0.0009) .P = 0.018), early (pT1–2) stage (P = 0.0004), absence of local recurrence (P = 0.03) and higher 5-year survival rate (P = 0.033) (High density of CD15+ infiltrating cells (≥46 cells/punch), detectable in 10.8% of tumors, was significantly associated with left sided location (= 0.033) .P<0.0001), whereas correlations of lesser significance were detectable with CD16+ (r = 0.32), CD68+ (r = 0.35), CD8+ (r = 0.13) and FOXP3+ (r = 0.21) cell infiltration.To explore relationships between tumor infiltration by cells expressing MPO and other immune markers  expressed by immunocompetent cells infiltrating CRC, data from additional immune-histochemical stainings of the same TMA from previous studies were used To assess in detail phenotypic characteristics of tissue infiltrating MPO+ cells, freshly excised CRC (n = 8) were enzymatically digested, and single cell suspensions were analyzed by flow cytometry. Examples of this phenotypic analysis are reported in The large majority of CRC infiltrating, MPO+ cells were CD15+ (90.3%±5.6%), CD16+, (77.6±19.4%), and CD66b+  associated with better prognosis , as compared to tumors with low MPO+ cell infiltration in univariate Cox regression analysis. Upon splitting of the cohort in a test and a validation set, high score MPO+ cell infiltration was still associated with significantly improved survival .Most importantly, in multivariate analysis, high score MPO+, but not CD15+, cell infiltration was independently associated with favorable prognosis after adjusting for several known prognostic factors such as age, sex, T stage, N stage, tumor grade, vascular invasion, tumor border configuration and microsatellite stability  consistent with a mature granulocyte nature of MPO+ infiltrating cells MPO activity has been suggested to contribute to the pathogenesis of degenerative diseases, including atherosclerosis, multiple sclerosis and Alzheimer disease Despite early reports documenting their ability to mediate tumor cell cytotoxicity More recently, evidence of the ability of granulocytes to inhibit lung metastasis formation in an experimental breast cancer model was provided The molecular background underlying CRC infiltration by MPO+ cells and its prognostic significance is unclear. These phenomena could reflect chemokine production by activated T cells, and therefore indirectly result from ongoing antitumoral adaptive responses. Alternatively, they might be related to the production of granulocyte attracting chemokines by tumor cells. At least CXCL8 (IL-8) is known to be produced by CRC cells. However, its production was suggested to be associated with increased angiogenesis and tumor dissemination Our results contribute to the characterization of the complex features inherent in gut microenvironment and with CRC-immune system interaction Within this framework, it is tempting to speculate that neutrophil infiltration should be included in current prognostic models for CRC


Salmonella Typhimurium causes the study of this zoonotic organism more important. Most studies on identification and characterization of S. Typhimurium are conducted at DNA level. Flagellin genes  are useful as a model system for studying genetic differentiation. The objectives of the present study were to identify the polymorphism of fljB among avians in different regions by the PCR-RFLP method.Economic constraint of diseases arising from S. Typhimurium isolates out of 1,870 intestine samples were identified using culture and serotyping as well as multiplex-PCR (broiler (n=13), layer (n=12), duck (n=5), goose (n=5), sparrow (n=8), canary (n=3), pigeon (n=5) and casco parrot (n=1)). Amplification of fljB gene was performed and amplified products subjected to restriction digestion with Hha I enzyme.Fifty-two Salmonella Typhimurium recovered from 13 broilers (ten with pattern A and 3 with pattern B) and 8 sparrow (three with pattern A and 5 with pattern B) showed both A and B patterns. Twelve layers, 5 pigeons and 3 canaries showed pattern A and 5 ducks, 5 geese and one casco parrot showed pattern B. None of these patterns was allotted for a special region.Two RFLP patterns generated DNA fragments between approximately 50 to 800 bps. Pattern A was observed in 33 (63.46%) and pattern B in 19 (36.54%) of isolates. fljB gene is highly conserved among avians in different geographical regions, suggesting not only the importance of fljB gene in survival of organism in different environmental conditions but also the relation between proteins encoded by fljB gene and serotyping scheme.The results of the present study showed that  Salmonella colonizes vertebrate hosts, with outcomes ranging from subclinical to systemic infection with high mortality.Members of the genus Salmonella consists of only two species: S.enterica and S.bongori. Salmonella enterica is divided into the following six subspecies: S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. Indica were collected from different geographical regions including North and Northwest of Iran. All samples were taken to the microbiology laboratory of Urmia Veterinary Faculty on ice to be refrigerated and processed in the same day and in sterile conditions. Each sample (1-2 gram) was enriched in Selenit broth tubes and incubated overnight at 37°C. A loop of each tube was streaked onto CHROMagar™ Salmonella medium  plates incubated at 37°C for 24 h. Suspected colonies ) were subcultured on Salmonella-Shigella agar medium  plates to obtain pure cultures. All pure isolates were Gram-stained and tested for their characteristic biochemical reactions.The bacteria were maintained on MacConkey agar plates. For longer storage, isolates grown in Leuria- Bertani broth were mixed with glycerol to obtain final concentration of 15% glycerol and kept at −80°C .Serotyping of isolates. All isolates presumptively identified as Salmonella, were serotyped at Razi vaccine and serum research institute (Iran) using slide agglutination test, and S. Typhimurium isolates were identified according to their serotyping formula: 1, 4,5, 12: i: 1, 2. Finally, a total of 52 S. Typhimurium were identified from various avians; broiler (n=13), layer (n=12), duck (n=5), goose (n=5), sparrow (n=8), canary (n=3), pigeon (n=5) and casco parrot (n= 1). To confirm the results of serotyping, all isolates were tested by multiplex-PCR.Genomic DNA extraction. DNA extraction of all Salmonella isolates were performed from overnight culture in buffered peptone water by Genomic DNA purification kit  with some modifications including the use of phenol-chloroform- isoamylalcohol (25: 24: 1) instead of chloroform which was indicated in DNA extraction protocol. Purity of DNA was tested spectrophotometrically at wave length of 260 and 280 nm . Extracted DNA was diluted to give a final concentration of 50 ng.Multiplex-PCR reaction for isolates. All isolates tested by multiplex-PCR have been described previously , dNTP (200 µM), primer (1 µM) and Smartaq™ DNA polymerase, (1U)  and template DNA (50 ng).eviously  with someviously . Four seeviously , includeFor negative control, sterile water was added instead of nucleic acid. PCR reaction was performed in a DNA thermocycler  as follows:an initial denaturation at 95°C for 5 min., 35 cycles of denaturation at 95°C for 1min, annealing at 65°C for 1min, elongation at 72°C for 30s and final 7min extension period at 72°C. Amplified products were separated by 2% agarose gel electrophoresis at 80 V for 1 h and photographed under UV illuminator.Amplification offljBgene. Amplification of fljB gene was performed by primer pair previously reported and used to amplify a fragment of approximately 1.5 kb , dNTP (400 µM), each primer (1 µM) and Smartaq™ DNA polymerase (2U)  and 50 ng of extracted DNA. For negative control, sterile water was added instead of nucleic acid. Amplification was performed in a DNA thermocycler  as follows:an initial denaturation at 95°C for 3 min, followed by 45 cycles of 1 min. at 94°C, 1 min at 58°C and 1 min at 72°C with a final step at 72°C for 5 min. The amplified products were visualized by gel electrophoresis using 10 µl of final reaction mixture on 0.8% agarose gel for 1 h at 100 V.Restriction digestion of PCR-amplifiedfljBgene. 10–15 µl of each PCR-amplified product was digested with 10 U of Hha I restriction enzyme at 37°C for 3 h according to manufacturer‘s instructions . Digested PCR products were separated by electrophoresis at 80V on 2% agarose gel for about 2h and photographed under UV illuminator.Identification ofS.Tyhpimurium. Out of 1, 870 samples, 52 were isolated as S. Tyhpimurium (broiler (n=13), layer (n=12), duck (n=5), goose (n=5), sparrow (n=8), canary (n=3), pigeon (n=5) and casco parrot (n=1)). The results of serotyping were confirmed with multiplex-PCR. Primers listed in table 1 for multiplex-PCR could successfully amplify the expected sizes: 183, 284, 526 and 663 bp from fliC, invA, fljB and rfbJ genes, respectively  and 8 sparrows (three of which pattern A and 5 pattern B) showed both A and B patterns, but 12 layers, 5 pigeons and 3 canaries showed pattern A and 5 ducks, 5 geese and one casco parrot showed pattern B  showed that an amplicon of fljB was observed for 100% of S. Typhimurium isolates, while 98% (47 of 48 isolates) of Salmonella serovar 4 [5] 12: i:- isolates produced no amplification. The results of serotyping and biochemical tests in their study could indicate that the isolate has a different mutation leading to the loss of the second-phase flagellar antigen  concluded that monophasic strains in which the phase-2 flagellar antigen is not detected serologically but can be detected by PCR may contain deletions in a part of fljB or fljB promoter controlling expression may become locked in one position that leave the H: 1, 2-specific PCR primer binding sites intact, or they may represent ‘serotype inconsistent’ strains  in 11 S. Typhimurium isolates corresponding to six phage types, but not in agreement with Jong et al. (2010) study which could be due to insufficient number of S. Typhimurium isolates (n=2). Dauga et al. (1998) demonstrated that endonuclease HphI showed fewer profiles than Hha I and fewer antigen or serovar-specific patterns than Hha I.Hsly used  for genefliC and fljB, encoding phase-1 and phase-2 flagellin of Salmonella enterica, offers a potential biomarker for Salmonella subtyping  and pattern A and pattern B were observed in both broilers and sparrows which showed close relation of flagellin genes in these host species. Sparrows nest in regions close to poultry farms and this may prepare the way for transmission of S. Typhimurium between these hosts. Ducks, geese and casco parrot together with pigeons and canaries showed identical RFLP patterns B and A, respectively. The plausible explanation for this may be due to the condition in which ducks and geese are bred closely, but it needs more isolates to compare, especially for casco parrot. We cannot allocate each unique pattern for a special host  and these patterns cannot show fljB gene characteristics in special host species because more isolates need to be tested.In the present study, the size of DNA in RFLP profile varied between 50bp to 800bp in profile B and in profile A was between 50 and 400bp. The variation of fljB gene in S. Typhimurium is highly conserved among different host species. This means that conserved region of this gene is vital for S. Typhimurium to protect it in different conditions, as it was mentioned before. The findings of present study in agreement with previous studies confirmed that flagellin genes (fljB gene in present study) encode proteins on Salmonella surface in relation to serotyping scheme. These conserved parts might be good candidates to develop vaccine against S. Typhimurium and other Salmonella which harbor the same conserved regions. This will be achieved through further studies on sequences of flagellin genes.In conclusion, the structure of


Colletotrichum graminicola was optimized using Response Surface Methodology (RSM). Maximal production occurred in wheat bran. Sugarcane trash, peanut hulls and corncob enhanced β-glucosidase, β-xylosidase and xylanase production, respectively. Maximal levels after optimization reached 159.3 ± 12.7 U g−1, 128.1 ± 6.4 U g−1 and 378.1 ± 23.3 U g−1, respectively, but the enzymes were produced simultaneously at good levels under culture conditions optimized for each one of them. Optima of pH and temperature were 5.0 and 65 °C for the three enzymes, which maintained full activity for 72 h at 50 °C and for 120 min at 60 °C (β-glucosidase) or 65 °C (β-xylosidase and xylanase). Mixed with Trichoderma reesei cellulases, C. graminicola crude extract hydrolyzed raw sugarcane trash with glucose yield of 33.1% after 48 h, demonstrating good potential to compose efficient cocktails for lignocellulosic materials hydrolysis.Efficient, low-cost enzymatic hydrolysis of lignocellulosic residues is essential for cost-effective production of bioethanol. The production of β-glucosidase, β-xylosidase and xylanase by  Recent years have seen an ever-increasing interest in cellulose and hemicellulose hydrolysis, in an effort to develop a cost-effective process to use lignocellulosic residues as raw materials for the production of value added products, mainly bioethanol –3. In BrThe enzymatic treatment is considered the most promising method for an efficient hydrolysis of cellulose in lignocellulosic residues ,8–10. HoDuring the last few years, researchers’ interest has been mostly focused on cellulases and cellulase-based cocktails for cellulose hydrolysis. However, it is now well known that enzymatic cocktails containing both cellulases and hemicellulases are required for an efficient saccharification of cellulose in lignocellulosic residues, resulting in higher yields of fermentable sugars with lower enzyme loads ,10,13,14exo-1,4-β-glucanases or cellobiohydrolases (EC 3.2.1.91), and 1,4-β-glucosidases (EC 3.2.1.21). Endoglucanases randomly cleave internal glycosidic linkages in cellulosic chains, while exoglucanases liberate cellobiose from their edges. β-glucosidases then act on celloligosaccharides and cellobiose, liberating glucose [Endo-β-1,4-xylanases (EC 3.2.1.8) randomly cleave β-1,4-glycosidic linkages in the xylan main chain, releasing xylooligosaccharides, while β-xylosidases (EC 3.2.1.37) release xylose from xylobiose and xylooligosaccharides. Like glucanases, xylanases are also usually inhibited by xylobiose and short xyloligosaccharides, and β-xylosidases are then responsible for the rate-limiting step of xylan hydrolysis. Accessory enzymes, including α-l-arabinofurosidase (EC 3.2.1.55), remove side groups from the xylan main chain [Cellulose enzymatic hydrolysis requires the synergistic action of endo-1,4-β-glucanases (EC 3.2.1.4), EC 3.2.1.1, and 1, glucose ,16. Owinin chain ,18.Most commercial cellulases and hemicellulases are currently produced by fungi under submerged fermentation. However, solid-state fermentation (SSF) is increasingly being considered an ideal, cost-effective technology for the production of cellulolytic enzymes. SSF conditions mimic the natural environment of fungi, resulting in higher fermentation productivity. Further, cellulases and hemicellulases produced under SSF are often more thermostable and pH-resistant ,19–21. OColletotrichum and their teleomorph Glomerella are recognized as one of the most important plant pathogens distributed on earth, particularly in tropical and subtropical regions. Colletotrichum causes plant diseases, usually known as antracnoses. Although different species from this genus are considered models for genetic and physiological studies [Colletotrichum graminicola was isolated from the Amazon soil, and preliminary studies revealed that it had good potential for the production of β-glucosidases, xylanases and β-xylosidases under SSF, but secreted low levels of cellulases. Thus, crude culture extracts from this strain, supplemented with cellulases, could be interesting for the hydrolysis of lignocellulosic residues, such as SCT.The filamentous fungi from the genus  studies , their pTrichoderma reesei is the best-known cellulase producer among filamentous fungi, and the preferred organism for the production of industrial cellulases [T. reesei has been extensively investigated. It produces good levels of two cellobiohydrolases (CBH1 and CBH2) and at least four endoglucanases , but low levels of two β-glucosidases (BGLI and BGLII). Thus, cellulose treatment with T. reesei crude culture extracts without an additional load of β-glucosidase results in the accumulation of cellobiose and consequent inhibition of endo- and exo-cellulases, with low glucose yields [llulases ,24. The e yields ,25.C. graminicola under SSF in low-cost media constituted of agroindustrial byproducts and/or residues. The efficiency of the crude culture extract from C. graminicola alone or in mixture with T. reesei crude cellulases for the hydrolysis of sugarcane trash without pre-treatment (RSCT) was also investigated.In this study we optimized the production of β-glucosidase, β-xylosidase and xylanase by a newly isolated strain of −1 dry substrate and the fungus showed vigorous growth in this substrate. Good enzyme levels were also obtained in peanut hulls, corn and rice husks. Although the fungus grew poorly in SCT, with a low enzyme production, the supplementation of wheat bran with 1% (w/w) SCT resulted in 1.6-fold increase in enzyme production levels. Enhanced production (1.4-fold) also resulted from wheat bran supplementation with 1% (w/w) cellobiose, but filter paper and carboxymethylcellulose (CMC) showed negligible effects. Addition of organic and inorganic nitrogen sources to wheat bran inhibited β-glucosidase production, except for yeast extract and soybean meal, without effect (The best carbon source for β-glucosidase production was wheat bran . The levt effect .−1), but milled corncob, soybean meal and corn husks were also acceptable sources for enzyme production 2SO4 and 1% peptone to wheat bran increased xylanase production , while all other nitrogen sources tested inhibited the production, except for casein  results in considerable increases in enzyme productivity by microorganisms. However, besides being expensive and time consuming, this methodology does not show the interactions that may occur between different variables that affect enzyme production. In contrast, statistical approaches using response surface methodology not only allow the identification of the optimal conditions by a set of independent variables, but also reveal the interactions between two or more variables .After preliminary studies by OFAT to define the experimental ranges for each independent variable, the culture conditions for the production of β-glucosidase, β-xylosidase and xylanase were optimized using a Central Composite Rotational Design (CCRD).Data obtained for each enzyme , 3 and 4x, y, z, w, k and s were the coded values for temperature, culture time, initial moisture, peanut hulls, milled corncob and (NH4)2SO4 concentrations, respectively.where w/w) in the culture medium enhanced the production of β-glucosidase by about 60%, compared to that obtained in wheat bran and water only (not shown). However, similar production was obtained for all SCT concentrations tested in this range. In agreement, when the production of β-glucosidase was optimized using RSM the concentration of SCT and its interactions with the other independent variables tested  has not significantly influenced the production (not shown). Thus, the concentration of SCT in the culture medium was fixed at 1% for further experiments. Similarly, the temperature and its interactions with the other independent variables tested have not significantly influenced the production of β-xylosidase (not shown), and the production of this enzyme was optimized with respect to culture time, initial moisture and peanut hulls concentration only.Preliminary analyses by OFAT revealed that the presence of SCT  for the response surface quadratic models for the production of each enzyme are summarized in P-values showed that the quadratic terms of all independent variables tested had significant effects on β-glucosidase production, but not their linear terms nor the interactions between them. The quadratic effects of culture time and initial moisture were the most important factors affecting β-glucosidase production.The regression coefficients obtained for β-glucosidase, β-xylosidase and xylanase production are listed in Significant effects on β-xylosidase production were exerted by culture time and initial moisture, the interaction between them, and the interaction between peanut hulls concentration and initial moisture. The quadratic terms of all three independent variables also had significant effects. The linear effects of culture time and initial moisture were the most critical variables that affected the production of β-xylosidase.P-values for the regression coefficients obtained for xylanase production revealed that temperature, culture time, initial moisture and (NH4)2SO4 concentration, and their quadratic terms, significantly affected the enzyme production. The quadratic term of milled corncob concentration and the interaction between temperature and initial moisture also had significant effects. The most important factors that affected xylanase production were the quadratic terms of temperature, culture time and initial moisture.Analysis of the Among the factors that influence the enzymatic production under SSF, water content is well recognized as one of the most critical. The ideal moisture varies with the organism, the operational conditions and the solid substrate, affecting both the microbial growth and the production and secretion of enzymes. At low moisture content, lower substrate swelling occurs, reducing the superficial area. Moreover, the solubility and diffusion of the nutrients are reduced, as well as the stability of the extracellular enzymes, possibly impairing the growth of the microorganism. In contrast, moisture levels that are too high may cause aggregation of substrate particles, reducing the porosity and thus limiting oxygen availability, also hindering microbial growth and lowering enzyme production ,33,34.i.e., its capability to bring the expected results in the presence of deviations from the optimal conditions or unexpected adverse factors. The 3D response curves for the most important factors that affect the production of β-glucosidase and β-xylosidase are shown in Tridimensional response surface curves graphically represent regression equations, demonstrating the relationships between the response and experimental levels of each variable and allowing a better understanding of the interactions between the significant independent variables. Moreover, they provide working ranges for each variable, depicting the robustness of a process, −1, using wheat bran as carbon source and 1% (w/w) sugarcane trash as supplementary carbon source. Maximum predicted value was 156.63 U g−1, 1.4-fold higher than observed before optimization. The mean value of the experimental validation of the optimized conditions (159.3 ± 12.7 U g−1) was in excellent correlation with the predicted value, confirming the validity of the model. Interestingly, the production of β-glucosidase under optimal temperature, initial moisture and culture time decreased about 50% in the absence of SCT, but near maximal levels were obtained with SCT concentrations ranging from 1% to 10% (w/w) (not shown). Considering that SCT does not support the growth of C. graminicola, it is possible that when grown in wheat bran, the fungus partially degrades SCT, liberating little amounts of some compounds that stimulate enzyme production.Statistical analysis showed that maximum β-glucosidase production could be achieved after 7.07 days at 28.13 °C, with initial moisture of 2.38 mL gw/w) and initial moisture of 2.1 mL g−1, at 25 °C, using wheat bran as carbon source. Maximum predicted production was 125.88 U g−1, about 2.2-fold higher than that obtained initially. The mean value of the experimental validation was 128.1 ± 6.4 U g−1, in good agreement with the predicted value, proving the model’s adequacy.For β-xylosidase production, the maximum point predicted was 7.71 days, peanut hulls concentration of 4.66%  and (NH4)2SO4 concentration of 0.378% (w/w), using wheat bran as carbon source. The maximal predicted value, 396.65 U g−1, was 2.1-fold higher than initially estimated, and showed good correlation with the result of the experimental validation (378.1 ± 23.3 U g−1), establishing the model’s validity.According to the model, maximum xylanase production occurred at 29.7 °C, 8.66 days, initial moisture of 2.21 mL gC. graminicola were higher than most reported in the literature for SSF processes, but values of the same magnitude were found for Aspergillus fumigatus [Trichoderma koningi F244 [Thermoascus aurantiacus grown in wheat straw [Aspergillus terreus M11 in corn stover [Aspergillus niger KK2 in rice straw supplemented with salts, yeast extract and corn steep liquor were similar to those found for C. graminicola [Aspergillus tamarii cultured in wheat bran supplemented with Vogel salts [Humicola lanuginosa M7D cultured in corncob supplemented with Vogel salts and corn steep liquor [C. graminicola, were produced by A. niger in wheat bran supplemented with mineral salts [A. niger and T. reesei [The optimal production levels of β-glucosidase by umigatus  and Tricngi F244  culturedat straw  and Aspen stover , both suminicola . Higher el salts  and for p liquor . Similaral salts . However. reesei .C. graminicola as a very promising microorganism for industrial purposes, since few fungal strains produce similar or higher levels of β-glucosidase, β-xylosidase and xylanase under SSF [Due to the high enzyme loads required for an efficient hydrolysis of lignocellulosic materials, it is estimated that enzyme costs contribute 23%–40% to cellulosic ethanol production cost, directly impacting the economic viability of the process ,42. The nder SSF ,35,38,40nder SSF . Thus, tC. graminicola cultured under optimal conditions were evaluated using a CCRD for each enzyme, and RSM analyses. The experimental conditions and results of the experimental designs are summarized in The effects of pH and temperature on the enzymatic activities in the crude extracts from F test, the models were predictive of the enzymatic activities as a function of pH and temperature, given the higher values for calculated F values compared to listed ones. The low p-values also indicate the models’ significance. Further, the p-values greater than 0.05 indicated that the lack of fit for the models was non-significant. The R2 coefficients of 0.99 for β-glucosidase, 0.99 for β-xylosidase and 0.98 for xylanase activities confirm the goodness of the models.The analyses of variance (ANOVA) for the response surface quadratic models are summarized in p-values for all linear and quadratic effects, and also for the interaction between pH and temperature, indicating their significant effect on enzymatic activities. The pH effects were higher than the temperature effects for the three activities, and working ranges for near optimal activity may be easily anticipated from the response surface curves for each enzymatic activity , confirming the model validity. β-xylosidase activity was maximal at pH 4.77 and 63.33 °C, with a predicted value of 32.59 U mg−1, in excellent correlation with the value of experimental validation (32.4 ± 0.8 U mg−1). Further, optimal pH and temperature for xylanase activity were 5.21 and 66.91 °C with a predicted value of 49.1 U mg−1, in close agreement with the experimental validation, 49.3 ± 1.4 U mg−1.The statistical analysis showed that optimum pH and temperature for β-glucosidase activity were 4.94 and 64.61 °C, and the maximum predicted value was 51.16 U mgC. graminicola enzymes are in the range found for most fungal β-glucosidases [pH optima determined for osidases ,44, β-xyosidases  and xylaosidases . MoreoveC. graminicola maintained about 100% of the initial activity up to 72 h at 50 °C. Moreover, the enzyme was fully stable for 120 min at 55–60 °C. At 65 °C, a rapid decrease to about 80% of the initial activity occurred after 10 min, but a residual activity of 63% remained after 120 min . Considering that the β-glucosidase, β-xylosidase and xylanase in C. graminicola crude extract showed optimal pH around 5.0, where they were fully stable at 50 °C up to 72 h and presented about 55% of their maximal activity at 50 °C, the conditions chosen for RSCT hydrolysis were 50 °C and pH 5.0.Initially, the optima of temperature and pH, and the thermal stability of the cellulases in the crude extract from T. reesei and C. graminicola were then characterized for specific activities at 50 °C and pH 5.0. Trichoderma reesei extract showed good levels of CMCase (3.7 ± 0.5 U mL−1), FPA (0.29 ± 0.04 U mL−1) and avicelase (0.30 ± 0.05 U mL−1), but low levels of cellobiase (0.002 U mL−1) and undetectable levels of the other activities tested. In contrast, low levels of cellulases were found in the extract from C. graminicola , which showed high levels of cellobiase (7.6 ± 0.9 U mL−1), β-xylosidase (5.0 ± 0.8 U mL−1) and xylanase (34.6 ± 4.9 U mL−1) activities, as well as some α-L-arabinofuranosidase activity (0.11 ± 0.02 U mL−1). Similar profiles were obtained for C. graminicola crude extracts produced at optimized conditions for β-glucosidase, β-xylosidase or xylanase production, despite some variation in the proportions of the main enzymes (not shown).The crude extracts from C. graminicola and T. reesei crude extracts is shown in C. graminicola extract only resulted in maximal yields of 11.3% reducing sugars and 4.4% glucose after 72 h. Higher yields were obtained using T. reesei extract, attaining 18.3% reducing sugars and 18.7% glucose. However, when using a mixture of both extracts, a synergic effect was observed, and yields of 24.6% reducing sugars and 25.5% glucose were attained just after 24 h. Moreover, maximal yields reached 37.6% reducing sugars and 33.1% glucose, after 48 h. The low yields of glucose obtained using C. graminicola crude extract reflect its low levels of cellulases. The inefficient hydrolysis of cellulose results in low production of cellobiose and, consequently, low glucose release by β-glucosidases, although present at high levels. The higher levels of reducing sugars compared to glucose thus possibly reflect the action of xylanases and β-xylosidases on RSCT, generating xyloligosaccharides and xylose. In contrast, the reducing sugars obtained using the cellulase-rich crude extract from T. reesei must derive from cellulose hydrolysis. Some glucose was also obtained, since this extract presents low levels of β-glucosidases, but the plateau of reducing sugars observed after 48 h suggests accumulation of cellobiose, inhibiting endo- and exo-glucanases, as reported by others [C. graminicola extract), as well as cellulases (T reesei extract), as proposed by other authors [The time course of RSCT hydrolysis using y others ,25. The  authors ,13,14. T authors ,63,64.Despite its increasing importance as a sustainable feedstock for cellulosic ethanol production, little information is available on SCT, and few authors have studied its enzymatic hydrolysis. Moreover, most studies reported were carried out using SCT submitted to different pre-treatments to increase the accessibility of cellulose and hemicelluloses to the enzymes ,42,63–65et al. [C. graminicola crude extract. Similar FPU loads (15 FPU g−1 RSCT) were used in both studies. Further, a CMCase load about 1.7-fold lower was employed in this study. In contrast, the cellobiase and xylanase loads were about 3-fold higher, and our cocktail also contained around 225 U g−1 of β-xylosidase activity, almost absent (1.4 U g−1) from the other cocktail. Thus, our higher glucose yield may be directly related to a higher efficiency of xylan degradation, resulting in an improved action of cellulases. Indeed, the xylan content of SCT is around 23% (w/w) [C. graminicola crude extract seems to be naturally very well suited for the hydrolysis of this particular lignocellulosic residue, with the addition of crude cellulases only.The higher efficiency of our cocktail for RSCT hydrolysis, compared to that used by Sant’Ana da Silva et al. , may be 3% (w/w) , and theetc.) using the C. graminicola cocktail were not yet optimized, increases in glucose yields are to be expected. Moreover, the addition of some other enzymes to the cocktail, such as ligninases, may increase the hydrolysis efficiency, as well as some mild, low-cost pre-treatment of SCT. Thus, this low-cost cellulolytic-hemicelulolytic enzyme system seems promising for future industrial use. However, its real practical applicability depends on several factors, including the success of scaling up the processes of enzyme production and SCT hydrolysis. Further, the implementation of any industrial process of enzymatic hydrolysis of a lignocellulosic residue for ethanol production depends on a complete techno-economic evaluation of the final production costs [Finally, considering that the conditions for the hydrolysis of RSCT  which contains a mixture of benzylpenicillin benzathine, benzylpenicillin procaine, diidroestreptomicin sulfate, potassium benzylpenicillin and streptomycin sulfate. The cultures were incubated at 25 °C for 48–72 h and the initial colonies formed were transferred and maintained in laboratory in PDA slant medium. Colletotrichum graminicola was classified on basis of physiological and morphological characteristics, including the conidiophores morphology, analyzed according to Barnett and Hunter [Trichoderma reesei QM9414 (ATCC 26921) was maintained at 25 °C on solid 4% oatmeal baby food (Quaker) medium.The d Hunter  and confd Hunter . The fun2) of the mycelium of C. graminicola grown on PDA medium were taken from 8-day-old cultures and inoculated into sterile solid media prepared in 250 mL Erlenmeyer flasks, containing 10 mL of deionized water and 5 g of different dry carbon sources. Each section contained fungi in all extension and was put onto the culture medium using an inoculation needle, in order to achieve the maximum contact between the mycelium and the medium. The transferences of the mycelial sections were performed in an inoculation chamber, and all the usual aseptic procedures were maintained both during the transferences and the cultures growth. The flasks were incubated for 192 h under static conditions in a controlled environment incubator  at 25 °C and 70% humidity, monitored by a thermo hygrometer . All experiments were conducted in triplicate. The enzymatic assays were carried out in duplicate. After choosing the best carbon source for the production of each enzyme, the effect of supplementation with different nitrogen and carbon sources was investigated under the same culture conditions.Sections  with 8 axial and 3 central points  which resulted in 27 experiments. The experimental ranges of each independent variable tested in RSM experiments were determined preliminarily, using the OFAT methodology, corresponding to a sugarcane trash concentration range of 0.5% to 10% (w/w), a temperature range of 20–40 °C, culture time from 7 to 11 days and medium initial moisture from 1.0 to 2.5 mL deionized H2O g−1 dry substrate . Since SCT concentration and its interactions with the other independent variables tested have not significantly influenced enzyme production, SCT was added to wheat bran at 1% (w/w) fixed concentration. The levels of the other independent variables  were then defined according to a 23 full-factorial central composite design (star configuration) with six axial and two central points  which resulted in 17 experiments  were defined according to a 2eriments .4 full-factorial central composite design (star configuration) with 8 axial and 3 central points  which resulted in 27 experiments. The experimental ranges of each independent variable tested were defined preliminarily using the OFAT methodology, corresponding to a peanut hulls concentration range of 0.5% to 7% (w/w), a temperature range of 20–40 °C, culture time from 6 to 10 days and medium initial moisture from 0.5 to 3.0 mL deionized H2O per g dry substrate . Since temperature and its interactions with the other independent variables tested have not significantly influenced enzyme production, it was fixed at 25 °C. The levels of the other independent variables  were defined according to a 23 full-factorial central composite design (star configuration) with six axial and three central points  which resulted in 17 experiments  were defined according to a 2eriments .5−1 fractional factorial central composite design (star configuration) with ten axial and six central points , resulting in 32 experimental runs , milled corncob concentration, 0.0%–2.0%, w/w; ammonium sulfate concentration, 0.1%–0.9% (w/w). All the experiments were conducted in triplicate and the enzymatic assays were carried out in duplicate. Initial moisture of the culture media was expressed as volume of deionized H2O (mL) per g dry substrate or as wet basis moisture content, experimentally determined and calculated according to the following equation:For xylanase production optimization, the levels of the independent variables  were defined according to a 2tal runs . The expMwb (%) = wet-basis moisture content; m2OH = mass of moisture (g), and mds = mass of dry substrate (g).where C. graminicola growth, culture medium was suspended in 30 mL cold water, gently homogenized with a glass rod in an ice bath, filtered through a nylon sieve and centrifuged at 10,000× g and 4 °C for 15 min. The supernatant (crude extract) was maintained at 4 °C with no appreciable losses of β-glucosidase, β-xylosidase and xylanase activities up to 30 days.After d-glucopyranoside and p-nitrophenyl-β-d-xylopyranoside , as described by Souza et al. [−1 sodium acetate buffer, pH 5.0, and 2.0 mmol L−1 substrate, in a final volume of 0.6 mL. Xylanase activity was routinely assayed in the same conditions using 1% (m/v) Birchwood xylan  as substrate, in a final volume of 1.0 mL. The reducing sugars released were quantified using the dinitrosalicylic acid (DNS) method [β-glucosidase and β-xylosidase activities were routinely determined using p-nitrophenyl-β-a et al. . Standar) method .C. graminicola and T. reesei employed in RSCT hydrolysis, β-glucosidase, β-xylosidase and xylanase activities were assayed as above, at 50 °C. α-l-Arabinofurosidase activity was assayed in the same conditions using p-nitrophenyl-α-L-arabinofuranoside  as substrate, according to the method described by Souza et al. [−1) was estimated under the same conditions by quantifying glucose released using the glucose oxidase method [®, Fluka Chemical Co.: Seelze, Germany), carboximethylcellulose (CMC) and filter paper (FPase activity) were assayed in the same conditions using Avicel or CMC at 1% (w/v) concentration, or a strip (1 cm × 3 cm) of Whatman No. 1 filter paper, quantifying the reducing sugars released by the DNS method.To characterize the crude extracts from a et al. . Hydrolye method . HydrolyExperimental conditions  employed in all activity measurements were adjusted to guarantee the estimation of initial velocities . One enzyme unit (1U) was defined as the amount of enzyme that releases 1 μmol of product per min. One filter paper enzyme unit (FPU) was defined as the amount of enzyme that releases 1 μmol of product per min using filter paper as substrate.C. graminicola crude extract the levels of the independent variables were defined according to a 22 full-factorial central composite design (star configuration) comprising 11 experimental runs, including 4 axial and 3 central points . The same design was applied for β-xylosidase and xylanase activities .RSM was conducted using the software Statistica, version 10.0  for the experimental designs and regression analyses of the data. A statistical analysis of each model was performed to estimate the analysis of variance (ANOVA), the quality of each polynomial model equation was statistically evaluated by the coefficient Three cultures for β-glucosidase, β-xylosidase and xylanase production, under optimized conditions, were carried out to validate the optimization. Similarly, the optimization of reaction pH and temperature for the enzymatic activities was validated by performing a triplicate experiment in the optimized conditions.Colletotrichum graminicola crude extract was obtained from 9-day cultures at 30 °C in wheat bran containing 0.9% (w/w) milled corncob and 0.4% (w/w) (NH4)2SO4, with initial moisture of 2.2 mL g−1 , at 70% humidity. Trichoderma reesei QM9414 was grown for 72 h at 28 °C and 130 rpm in 500-mL Erlenmeyer flasks containing 100 mL of liquid medium composed of 0.03% urea, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 5.0 mg mL−1 FeSO4·7H2O, 1.6 mg mL−1 MnSO4, 1.4 mg mL−1 ZnSO4. 7H2O, 2 mg mL−1 CoCl2, 0.075% peptone, 0.2% Tween-80 and 0.75% Avicel®, with initial pH adjusted for 5.5–6.0. Cellulase-rich T. reesei crude extract, obtained by filtration of the culture medium through synthetic sponge, was maintained at 4 °C for a month without appreciable activity losses.−1 sodium acetate buffer, pH 5.0, containing 1% (w/v) RSCT and 10 mmol L−1 sodium azide in a final volume of 20 mL, in hermetically closed 250-mL Erlenmeyer flasks. Hydrolysis was initiated by addition of an aliquot of T. reesei crude extract, an aliquot of C. graminicola crude extract or a mixture of both. After desired time intervals, 0.5 mL aliquots of the reaction medium were withdrawn, centrifuged at 10,000× g for 5 min and the supernatants were analyzed for reducing sugars and glucose.Before hydrolysis, RSCT  was washed until detection of negligible levels of reducing sugars in washing waters (DNS method). Washed RSCT was dried overnight at 60 °C and grinded with a domestic blender . Enzymatic hydrolysis was carried out at 50 °C and 180 rpm in 50 mmol LThe yield of RSCT hydrolysis was calculated as a percentage of maximal reducing sugars or glucose released by sulfuric acid hydrolysis, according to the National Renewable Energy Laboratory (NREL) Laboratory Analytical Procedure (LAP) for the quantification of structural carbohydrates .Colletotrichum graminicola is a significant producer of β-glucosidase, β-xylosidase and xylanase under SSF in cheap media, comprising of wheat bran as carbon source and agricultural residues as supplements. All three enzymes are produced simultaneously at good levels, and crude culture extracts may thus be used without further processing to compose efficient cocktails for lignocellulosic material hydrolysis. Indeed, a cocktail composed of C. graminicola crude extract supplemented with T. reesei crude cellulases hydrolyzed raw sugarcane trash with a yield of 33.1% glucose after 48 h, demonstrating strong potential for the hydrolysis of this interesting, cheap feedstock for the production of cellulosic ethanol.