Biomedical Sciences and Technology: Peer-Reviewed Journal Articles

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12521/723

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Spatial Organization and Recruitment of Non-Specific T Cells May Limit T Cell-Macrophage Interactions Within Mycobacterium Tuberculosis Granulomas
(Frontiers in Immunology, 2021) Butler, J. Russell
Tuberculosis (TB) is a worldwide health problem; successful interventions such as vaccines and treatment require a 2better understanding of the immune response to infection with Mycobacterium tuberculosis (Mtb). In many infectious diseases, pathogen-specific T cells that are recruited to infection sites are highly responsive and clear infection. Yet in the case of infection with Mtb, most individuals are unable to clear infection leading to either an asymptomatically controlled latent infection (the majority) or active disease (roughly 5%–10% of infections). The hallmark of Mtb infection is the recruitment of immune cells to lungs leading to development of multiple lung granulomas. Non-human primate models of TB indicate that on average <10% of T cells within granulomas are Mtb-responsive in terms of cytokine production. The reason for this reduced responsiveness is unknown and it may be at the core of why humans typically are unable to clear Mtb infection. There are a number of hypotheses as to why this reduced responsiveness may occur, including T cell exhaustion, direct downregulation of antigen presentation by Mtb within infected macrophages, the spatial organization of the granuloma itself, and/or recruitment of non-Mtb-specific T cells to lungs. We use a systems biology approach pairing data and modeling to dissect three of these hypotheses. We find that the structural organization of granulomas as well as recruitment of non-specific T cells likely contribute to reduced responsiveness.
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Does Teaching About Evolution in an Adventist School Destroy Faith in God?
(The Journal of Adventist Education, 2021) Dobias, Stanley D.; Butler, J. Russell
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Systems Biology Predicts that Fibrosis in Tuberculous Granulomaas May Arise Through Macrophage-to-Myofibroblast Transformation
(PLOS Computational Biology, 2020) Butler, J. Russell
Mycobacterium tuberculosis (Mtb) infection causes tuberculosis (TB), a disease characterized by development of granulomas. Granulomas consist of activated immune cells that cluster together to limit bacterial growth and restrict dissemination. Control of the TB epidemic has been limited by lengthy drug regimens, antibiotic resistance, and lack of a robustly efficacious vaccine. Fibrosis commonly occurs during treatment and is associated with both positive and negative disease outcomes in TB but little is known about the processes that initiate fibrosis in granulomas. Human and nonhuman primate granulomas undergoing fibrosis can have spindle-shaped macrophages with fibroblast-like morphologies suggesting a relationship between macrophages, fibroblasts, and granuloma fibrosis. This relationship has been difficult to investigate because of the limited availability of human pathology samples, the time scale involved in human TB, and overlap between fibroblast and myeloid cell markers in tissues. To better understand the origins of fibrosis in TB, we used a computational model of TB granuloma biology to identify factors that drive fibrosis over the course of local disease progression. We validated the model with granulomas from nonhuman primates to delineate myeloid cells and lung-resident fibroblasts. Our results suggest that peripheral granuloma fibrosis, which is commonly observed, can arise through macrophage-to-myofibroblast transformation (MMT). Further, we hypothesize that MMT is induced in M1 macrophages through a sequential combination of inflammatory and anti-inflammatory signaling in granuloma macrophages. We predict that MMT may be a mechanism underlying granuloma-associated fibrosis and warrants further investigation into myeloid cells as drivers of fibrotic disease.
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Tissue Factor-dependent and -independent Pathways of Systemic Coagulation Activation in Acute Myeloid Leukemia: A Single-center Cohort Study
(Experimental Hematology & Oncology, 2015) Amirkhosravi, Ali
Background In acute myeloid leukemia (AML), disseminated intravascular coagulation (DIC) contributes to morbidity and mortality, but the underlying pathomechanisms remain incompletely understood. Methods We conducted a prospective study on 69 patients with newly diagnosed AML to further define the correlates of systemic coagulation activation in this hematological malignancy. Tissue factor procoagulant activity (TF PCA) of isolated peripheral blood mononuclear cells (PBMCs) and TF expression by circulating microparticles (MPs) were assessed by single-stage clotting and thrombin generation assay, respectively. Soluble plasma TF antigen and secretion of vascular endothelial growth factor (VEGF) by cultured PBMCs were measured by ELISA. Cell-free plasma DNA was quantified by staining with a fluorescent dye. Result TF PCA of PBMCs was significantly increased in AML patients as compared to healthy controls. Furthermore, TF PCA was significantly associated with decompensated DIC at presentation, as defined by a plasma fibrinogen level of ≤1 g/L (n = 11). In addition to TF PCA and circulating blasts, serum lactate dehydrogenase, a surrogate marker for leukemic cell turnover, correlated with plasma D-Dimer in the total patient cohort and was significantly increased in DIC patients, suggesting a role for myeloblast apoptosis/necrosis in activation of the TF-dependent coagulation pathway. Consistently, TF-bearing plasma MPs were more frequently detected and levels of soluble TF antigen were significantly higher in DIC vs. non-DIC patients. No association was found between TF PCA expression and VEGF secretion by isolated PBMCs, but significantly increased levels of cell-free plasma DNA pointed to a contribution of the intrinsic contact pathway to systemic coagulation activation in the total patient cohort and in patients with lower TF PCA expression. While PBMC-associated TF PCA had no effect on long-term survival, DIC occurrence at presentation increased the risk of early mortality. Conclusion In newly diagnosed AML, TF expression by PBMCs and shedding of TF-bearing plasma MPs are central to the pathogenesis of DIC, but additional pathways, such as DNA liberation, may contribute to systemic coagulation activation.
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CDP7657, an Anti- CD40L Antibody Lacking an Fc Domain, Inhibits CD40L-dependent Immune Responses Without Thrombotic Complications: An In Vivo Study
(Arthritis Research and Therapy, 2015) Amirkhosravi, Ali
Introduction CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8. Methods The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE. Results CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria. Conclusions These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.
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CD32a Antibodies Induce Thrombocytopenia and Type II Hypersensitivity Reactions in FCGR2A Mice
(Blood, 2015-11-05) Amirkhosravi, Ali
The CD32a immunoglobulin G (IgG) receptor (Fcγ receptor IIa) is a potential therapeutic target for diseases in which IgG immune complexes (ICs) mediate inflammation, such as heparin-induced thrombocytopenia, rheumatoid arthritis, and systemic lupus erythematosus. Monoclonal antibodies (mAbs) are a promising strategy for treating such diseases. However, IV.3, perhaps the best characterized CD32a-blocking mAb, was recently shown to induce anaphylaxis in immunocompromised “3KO” mice. This anaphylactic reaction required a human CD32a transgene because mice lack an equivalent of this gene. The finding that IV.3 induces anaphylaxis in CD32a-transgenic mice was surprising because IV.3 had long been thought to lack the intrinsic capacity to trigger cellular activation via CD32a. Such an anaphylactic reaction would also limit potential therapeutic applications of IV.3. In the present study, we examine the molecular mechanisms by which IV.3 induces anaphylaxis. We now report that IV.3 induces anaphylaxis in immunocompetent CD32a-transgenic “FCGR2A” mice, along with the novel finding that IV.3 and 2 other well-characterized CD32a-blocking mAbs, AT-10 and MDE-8, also induce severe thrombocytopenia in FCGR2A mice. Using recombinant variants of these same mAbs, we show that IgG “Fc” effector function is necessary for the induction of anaphylaxis and thrombocytopenia in FCGR2A mice. Variants of these mAbs lacking the capacity to activate mouse IgG receptors not only failed to induce anaphylaxis or thrombocytopenia, but also very potently protected FCGR2A mice from near lethal doses of IgG ICs. Our findings show that effector-deficient IV.3, AT-10, and MDE-8 are promising candidates for developing therapeutic mAbs to treat CD32a-mediated diseases.
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A Therapeutic Antibody-Drug Conjugate Targeting Tissue Factor with Potent Activity Against a Broad Range of Solid Tumors
(Cancer Research, 2014-02-15) Amirkhosravi, Ali
Tissue factor (TF) is aberrantly expressed in solid cancers and is thought to contribute to disease progression through its procoagulant activity and its capacity to induce intracellular signaling in complex with factor VIIa (FVIIa). To explore the possibility of using tissue factor as a target for an antibody-drug conjugate (ADC), a panel of human tissue factor–specific antibodies (TF HuMab) was generated. Three tissue factor HuMab, that induced efficient inhibition of TF:FVIIa-dependent intracellular signaling, antibody-dependent cell-mediated cytotoxicity, and rapid target internalization, but had minimal impact on tissue factor procoagulant activity in vitro, were conjugated with the cytotoxic agents monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). Tissue factor–specific ADCs showed potent cytotoxicity in vitro and in vivo, which was dependent on tissue factor expression. TF-011-MMAE (HuMax-TF-ADC) was the most potent ADC, and the dominant mechanism of action in vivo was auristatin-mediated tumor cell killing. Importantly, TF-011-MMAE showed excellent antitumor activity in patient-derived xenograft (PDX) models with variable levels of tissue factor expression, derived from seven different solid cancers. Complete tumor regression was observed in all PDX models, including models that showed tissue factor expression in only 25% to 50% of the tumor cells. In conclusion, TF-011-MMAE is a promising novel antitumor agent with potent activity in xenograft models that represent the heterogeneity of human tumors, including heterogeneous target expression.
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CalDAG-GEFI Deficiency Protects Mice from FcγRIIa-mediated Thrombotic Thrombocytopenia Induced by CD40L and β2-GPI Immune Complexes
(Journal of Thrombosis and Haemostasis, 2014) Amirkhosravi, Ali
Introduction Platelet activation via the Fcγ receptor IIa (FcγRIIa) is implicated in the pathogenesis of immune complex (IC)‐mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or β2 Glycoprotein I (β2GPI) induce ITT in mice transgenic for human FcγRIIa (hFcR) but not wild‐type controls (which lack FcγRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG‐GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs. Methods Pre‐formed anti‐CD40L or anti‐β2GPI ICs were injected into hFcR/Caldaggef1+/+ or hFcR/Caldaggef1−/− mice, with or without clopidogrel pretreatment. Animals were observed for symptoms of shock for 30 min, during which time core body temperature was monitored. Platelet counts were obtained before and 30 min after IC injection. Lungs were assessed for thrombosis by histology or near‐infrared imaging. Results Both CD40L and β2GPI ICs rapidly induced severe thrombocytopenia, shock and a reduction in body temperature in hFcR/Caldaggef1+/+ mice. hFcR/Caldaggef1−/− mice were protected from CD40L and β2GPI IC‐induced thrombocytopenia and shock, whereas P2Y12 inhibition had only a modest effect on IC‐induced ITT. Consistent with these findings, IC‐induced integrin activation in vitro and the accumulation of activated platelets in the lungs of IC‐challenged mice was strongly dependent on CalDAG‐GEFI. Conclusions Our studies demonstrate that CalDAG‐GEFI plays a critical role in platelet activation, thrombocytopenia and thrombosis induced by clinically relevant ICs in mice. Thus, CalDAG‐GEFI may be a promising target for the intervention of IC‐associated, FcγRIIa‐mediated thrombotic conditions.
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Microparticle Association and Heterogeneity of Tumor-derived Tissue Factor in Plasma: Is It Important for Coagulation Activation?
(Journal of Thrombosis and Haemostasis, 2014) Amirkhosravi, Ali
Background Tumor‐derived tissue factor (TF) activates coagulation in vitro and in vivo in an orthotopic model of human pancreatic cancer. Here, we further characterized tumor‐derived TF in this model. Methods Conditioned medium (CM) of L3.6pl human pancreatic tumor cells and plasma from nude mice bearing L3.6pl tumors were ultracentrifuged, and the pellets were filtered through membranes with different pore sizes. The size distribution of particles was analyzed in CM or plasma fractions with nanoparticle tracking and dynamic light scattering. Human TF antigen and activity were measured in pellets and supernatants with ELISA and clotting or thrombin generation assays, respectively. Human alternatively spliced TF (asTF) was measured with ELISA. Human TF and thrombin–antithrombin complex (TAT) concentrations were assessed in plasma of mice injected with filtered fractions of CM. Results Particles in both CM and plasma were < 0.4 μm. TF antigen and activity in the CM were mainly associated with microparticles (MP). Approximately 50% of antigen and 20% of activity were associated with particles of < 0.1 μm. Injection of < 0.1‐μm particles into mice caused a 30% drop in platelet counts and an increase in TAT levels. In contrast, ~ 90% of TF antigen in tumor‐bearing mice plasmas was non‐sedimentable, whereas TF activity was exclusively associated with MP. Particles of < 0.1 μm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. Although asTF was found in MP‐free CM supernatants, it was also present in CM and plasma pellets. Conclusions Tumor‐derived particles of < 0.1 μm and non‐sedimentable TF are or can become procoagulant in the presence of phospholipids, and may contribute to the procoagulant potential of circulating TF.
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Reducing Cardiovascular Risk in Women With Lupus: Perception of Risk and Predictors of Risk-reducing Behaviors
(Journal of Cardiovascular Nursing, 2014) Amirkhosravi, Ali
Background: Women with systemic lupus erythematosus (SLE) display a 7- to 10-fold increased risk for cardiovascular disease (CVD) compared with non-SLE controls, yet many are unaware of this risk despite years spent in the healthcare system. It is not clear why they lack awareness of increased CVD risk or which factors influence awareness. Objective: The purpose of this study was to assess in women with SLE their perceived CVD risk, the association between clinically identified and perceived CVD risk factors, and factors that influenced CVD risk awareness and adoption of risk-reducing behaviors. Methods: Questionnaires, face-to-face meetings, and clinical assessments were used to collect data on demographics, perceived CVD risk, perceived CVD risk factors, actual CVD risk factors, risk-reducing behaviors, and healthcare provider counseling from 60 women with SLE. Regression analyses identified factors that influenced risk awareness and adoption of risk-reducing behaviors. Results: Two-thirds of the participants perceived themselves at increased CVD risk when compared with women without SLE, but the same number did not perceive an increase in their absolute CVD risk. Age was a significant predictor ( P = .05) for awareness of increased absolute risk; younger age correlated with increased awareness. Most women received information about heart disease from public media. On average, participants had 4 CVD risk factors but perceived that they had only 2. Age ( P = .001) and the number of perceived risk factors ( P = .004) predicted adoption of risk-reducing behaviors ( P = .03). Conclusion: Participants underestimated their CVD risk factors and did not personalize their increased CVD risk. Healthcare providers’ identification and discussion of CVD risk factors in women with SLE may enhance their CVD risk awareness and the adoption of risk-reducing behaviors.
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Standardization of Microparticle Enumeration Across Different Flow Cytometry Platforms: Results of a Multicenter Collaborative Workshop
(Journal of Thrombosis and Haemostasis, 2017) Amirkhosravi, Ali
Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC‐optimized instruments, was recently proposed to reproducibly enumerate platelet‐derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet‐free plasma samples. The inter‐laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side‐scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead‐based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MPs’ clinical relevance at a multicenter level.
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Aflibercept Exhibits VEGF Binding Stoichiometry Distinct from Bevacizumab and Does Not Support Formation of Immune-like Complexes
(Angiogenesis, 2016) Amirkhosravi, Ali
Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.
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Microvesicle-associated Tissue Factor Procoagulant Activity for the Preoperative Diagnosis of Ovarian Cancer
(Thrombosis Research, 2016) Amirkhosravi, Ali
Background Tissue factor (TF) is involved in tumor growth and metastasis and contributes to venous thromboembolism (VTE) in cancer, including gynecological malignancies. The diagnostic value of microvesicle-associated TF procoagulant activity (MV TF PCA) in women with suspected ovarian cancer, however, has not been studied. Objective To evaluate MV TF PCA as a diagnostic tool in women with an ovarian mass of unknown etiology and as a predictive biomarker for perioperative VTE. Methods Plasma MVs were isolated by high-speed centrifugation and analyzed for TF-specific PCA by single-stage clotting assay. In addition, plasma TF antigen and soluble P-selectin (sCD62P) were measured by ELISA. Results D-Dimer, MV TF PCA, and sCD62P, but not the tumor marker, CA-125, significantly differentiated patients with malignant (n = 40) from those with benign tumors (n = 15) and healthy controls (n = 34). In cancer patients, only D-Dimer and CA-125 correlated with the FIGO stage. An abnormal D-dimer had the highest sensitivity for the diagnosis of cancer, while MV TF PCA above the ROC curve-derived cut-off value of 182 U/mL had the highest specificity. By multivariate logistic regression analysis, addition of MV TF PCA conferred diagnostic benefit to the single variables, CA-125 (p = 0.052) and D-dimer (p = 0.019). Perioperative VTE occurred in 16% of cancer patients and was associated with an advanced FIGO stage, but not MV TF PCA. There was no difference in plasma TF antigen levels between study groups. Conclusions MV TF PCA, but not plasma TF antigen, may provide valuable additional information for the diagnostic work-up of women with suspected ovarian cancer.
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Functional Characterization of a VEGF-A-targeting Anticalin, Prototype of a Novel Therapeutic Human Protein Class
(Angiogenesis, 2016) Amirkhosravi, Ali
Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood–retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.
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Dietary Pentosanase Supplementation of Diets Containing Different Qualities of Wheat on Growth Performance and Metabolizable Energy of Turkey Poults
(2004) Santos, Anael A. Jr
Wheat varies in apparent metabolizable energy N-corrected (AMEn) due to the presence of non-starch polysaccharides (NSP), which can be improved by dietary enzyme supplementation. Poults from 0-17 d-age were fed diets containing various wheat sources (WS) with or without Natugrain Blend® (NB) (BASF, Germany). Five replicate cages of 10 poults were assigned to each eight-soybean-meal/wheat treatment diets and a control soybean-meal/corn diet. The treatments were a factorial arrangement of 4 WS (A, B, C, D) and 2 enzyme levels (0 and 200 mg NB/kg). The WS differed by the degree of frost damage during seed development. Regardless of the source of wheat, NB increased 17 d BW (351 vs 381 g, P < 0.001), decreased 1-17 d FCR (1.55 vs 1.49, P < 0.05), increased AMEn (2,204 vs 2,455 kcal/kg, P < 0.001), and increased apparent nitrogen retention (ANR) (35.0 vs 41.4 %, P < 0.05). No effects of WS were seen on growth performance, but WS A and B had higher (P < 0.05) AMEn than sources C and D (2,396 and 2,460 vs 2,246 and 2,216 kcal/kg, respectively). Gut viscosity was higher (P < 0.05) in poults fed wheat-based diets than the control diet. Enzyme supplementation to the wheat-based diets decreased viscosity (5.57 vs 3.98 cP, P < 0.05) to a level similar to the corn-based control diet, and it resulted in equivalent growth performance. Viscosities were negatively correlated with AMEn. The results demonstrated a positive effect of enzyme supplementation on nutrient utilization and performance of turkeys.
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Dietary Supplementation of Endoxylanases and Phospholipase for Turkeys Fed Wheat-based Rations
(2004) Santos, Anael A. Jr
The adverse effects of non-starch polysaccharides (NSP) on turkeys fed wheat-based diets may be alleviated by dietary supplementation of endoxylanase (to reduce the adverse effects of digesta viscosity) or phospholipase (to improve the digestibility of fat). BUTA toms were fed wheat-based diets containing one of 5 enzyme treatments: unsupplemented control, Natugrain Blend® (> 5,500 EXU/kg diet; NB), Lyxasan®-50 (> 2,250 EXU/kg diet; LX50), Lyxasan®-100 (> 5,500 EXU/kg diet; LX100), and Phospholipase (> 500 PLU/kg diet; PL) (BASF, Germany). Each treatment group was assigned to 8 pens containing 12 birds to evaluate growth performance (1-128 d), and 2 pens of 12 birds (excluding LX50) for the apparent metabolizable energy N-corrected (AMEn) and ileum viscosity determination (56-128 d). All enzyme treatments improved growth performance. In comparison to the control, dietary enzyme increased (P < 0.05) BW and decreased 1-128 d feed/gain (2.45 vs 2.37, P < 0.005). PL was most effective in reducing feed/gain during the starting phase and LX100 during the finishing phase, while NB had intermediate benefits throughout the experiment. PL increased AMEn from 9 to 12 wk, while NB and LX-100 resulted in the highest AMEn during the later finishing period. Viscosity was significantly higher for PL than the other treatments (13.5 vs 7.07 cP, P < 0.001). Growth performance and energy utilization of turkeys fed wheat-based diets can be significantly enhanced by phospholipase supplementation of starter feeds and endoxylanase supplementation of growing and finishing feeds. However, enzyme blends may provide a positive response regardless of turkey age.
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Influence of Grain Particle Size and Insoluble Fiber Content on Salmonella Colonization and Shedding of Turkeys Fed Corn-Soybean Meal Diets
(2006) Santos, Anael A. Jr
This study aimed to determine the impact of feeding partially ground corn or insoluble fiber on intestinal development, Salmonella cecal colonization and fecal shedding of turkeys from 0-28d. Turkeys reared in cage-batteries were assigned to 1 of 3 diets: ground corn-SBM (GC, TRT 1), coarse ground corn-SBM (CC, TRT 2), and 4% wood shavings + ground corn-SBM (SC, TRT 3). A 3-strain cocktail of nalidixic acid-resistant Salmonella enterica serotypes Hadar, Javaina, and Typhimurium was orally-gavaged into each poult at placement. Cecal and fecal Salmonella populations, growth performance and intestinal weights and lengths were measured. The diets had no impact on Salmonella cecal or fecal populations. At 28d, Salmonella cecal populations decreased approximately 3-logs (range: 2.4-3.3 log reduction) across all treatments in comparison to 7d (P< 0.0001). At 28d body weight, body gain and feed conversion ratio were not impacted by the diets. However, at 14d poults consuming the SC diet had lower feed consumption than those fed the GC and CC diets (231 vs. 243 and 252 g, P=0.001, respectively). The CC diet resulted in heavier relative gizzard weights at 28d in comparison to the GC and SC diets (30 vs. 28 and 22 g/kg, respectively, P< 0.0001). Conversely, the SC treatment reduced the mass of the small intestine relative to body weight, especially the jejunum. Dietary inclusion of coarsely ground corn and wood shavings had no adverse effect on growth performance yet improved gizzard and intestinal development, which could have positive effects on intestinal health.
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Determination of Ileum Microbial Diversity of Broilers Fed Triticale- or Corn-based Diets and Colonized by Salmonella
(2007) Santos, Anael A. Jr
Diversity of the bacterial communities in the ileum of broilers was characterized using denaturing gradient gel electrophoresis. Denaturing gradient gel electrophoresis separation of polymerase chain reaction amplicons of the V2–V3 variable regions of the 16S rDNA is a common method to profile community diversity and has been used to assess the effects of diet and antibiotics on the ileal bacterial community of chickens. Broilers raised either on litter floor or in cage batteries were fed either a finely ground corn- (control), a finely ground triticale-, or a whole triticale-based diet from 0 to 42 d. Microbial DNA was extracted from the ileum content of 42-d-old broilers, and the 16S rDNA gene was amplified by polymerase chain reaction and the amplicons separated by denaturing gradient gel electrophoresis. Diversity indexes including richness, evenness, diversity, and pairwise similarity coefficients were calculated. Diversity indexes were related to the dietary treatments, housing designs, and to changes in Salmonella colonization of broiler ceca as characterized by the most probable number method. Higher microbial diversity indexes were observed among birds fed whole triticale-based diets and reared on litter floors. In contrast, finely ground grain treatments had lower diversity and higher Salmonella prevalence than the whole triticale treatment. The data indicated that combination of high dietary fiber content and increased coarseness of the diet by feeding whole triticale stimulated microbial community diversity and discouraged Salmonella colonization, perhaps through a competitive exclusion-type mechanism.
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Influence of Housing System, Grain Type and Particle Size on Salmonella Colonization and Shedding of Broilers Fed Triticale or Corn-Soybean Meal Diets
(2008) Santos, Anael A. Jr
Salmonella colonization in poultry may be influenced by grain type and particle size. Broilers reared either in nonlitter cage-based housing or in a conventionally floored litter house from 0 to 42 d were assigned to 1 of 4 dietary treatments: 1) ground corn-soybean meal (C, 560 μm), 2) coarsely ground corn-soybean meal (CC, >1,700 μm), 3) ground triticale-soybean meal (T, 560 μm), or 4) whole triticale-soybean meal (WT). A 4-strain cocktail of Salmonella enterica was orally gavaged into each chick at placement. Growth performance, cecal and fecal Salmonella populations, gizzard and proventriculus pH, intestinal size, jejunum histomorphometry, and carcass yields were measured. Broilers responded differently to the dietary treatments according to the housing system used. At 42 d, birds reared on litter and fed ground grain had greater BW than those fed coarse grain (2.87 vs. 2.71 kg), whereas cage-reared broilers fed ground triticale were heavier than those fed corn (2.75 vs. 2.64 kg). Broilers raised on litter had a better feed conversion ratio than those raised in cages (1.71 vs. 1.81 g/g). Independent of the housing system, relative eviscerated carcass weights of birds fed T and C were heavier than those of CC- and WT-fed broilers (762 vs. 752 g/kg). Generally, the jejunum villus area and mucosal depth were larger, whereas the small intestine was lighter and shorter in broilers raised on litter. Relative gizzard weights of broilers raised on litter and fed the coarser diets were heavier than those of broilers reared in cages and fed finely ground diets. Feeding whole or coarsely ground grains decreased cecal Salmonella populations in 42-d-old broilers (3.8, 3.9, 4.4, and 4.4 log most probable number/g for CC, WT, C, and T, respectively). Additionally, 42-d-old broilers reared on litter had lower cecal Salmonella populations than those in cages (3.8 vs. 4.4 log most probable number/g). In conclusion, as a feed ingredient, triticale is a good alternative to corn, resulting in improved BW and reduced Salmonella colonization. Broilers raised on litter may have achieved lower cecal Salmonella populations than caged birds because access to litter may have modulated the intestinal microflora by increasing competitive exclusion microorganisms, which discouraged Salmonella colonization.
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Change in the Ileum Bacterial Population of Turkey Fed Different Diets and after Infection with Salmonella as Setermined with Denaturing Gradient Gel Electrophoresis Analysis of Amplified 16S Ribosomal DNA
(2008) Santos, Anael A. Jr
Changes in ileal bacterial populations of Salmonella-infected turkeys fed different diets were analyzed by using 16S-V3 PCR denaturing gradient gel electrophoresis. Turkeys raised on litter flooring were fed wheat- and corn-based diets with and without enzyme preparations (XY1 and XY2, respectively) from 0 to 126 d. Preparation XY1 contained exclusively endoxylanase, whereas preparation XY2 contained endoxylanase, protease, and α-amylase (Danisco, Wiltshire, UK). The dietary activity levels of XY1 and XY2 were 2,500 and 650 endo-1,4-β-xylanase units/kg of feed, respectively. Microbial DNA was extracted from the ileal content of 16-wk-old turkeys, and the 16S rDNA gene was amplified by PCR and analyzed by denaturing gradient gel electrophoresis. Diversity indexes, including richness (number of species, S), evenness (relative distribution of species, EH), diversity (using Shannon's index, H′), and Sorenson's pairwise similarities coefficient (measures the species in common between different habitats, Cs) were calculated. Additionally, diversity indexes were associated with Salmonella prevalence determined from fresh fecal droppings collected from each pen. On the basis of contrast analysis, the wheat-based diets resulted in higher microbial diversity indexes than the corn-based diets (S = 10 vs. 12; EH = 0.9 vs. 0.8; H′ = 2.2 vs. 1.9, P < 0.05). Likewise, enzyme supplementation stimulated growth of the microbiota and increased the diversity indexes in comparison with unsupplemented treatments (S = 13 vs. 10; EH = 0.9 vs. 0.8; H′ = 2.2 vs. 1.9, P < 0.05). Salmonella prevalence was higher (P < 0.05) at 15 wk in turkeys fed the corn-based diet (Salmonella prevalence = 50%) than in turkeys fed the corn-enzyme (Salmonella prevalence = 13%) and wheat-based (Salmonella prevalence = 0%) dietary treatments. Therefore, contrast analysis showed that birds fed the corn control diet had lower microbiota diversity but higher Salmonella prevalence than birds fed the enzyme-supplemented and wheat-based diets. In contrast, birds fed the wheat-based diets had higher diversity but lower Salmonella prevalence than birds fed the corn-based diets. High dietary nonstarch polysaccharides from wheat and dietary exogenous enzyme supplementation promoted microbial community diversity and apparently discouraged Salmonella colonization through competitive exclusion. Nonstarch polysaccharides and dietary exogenous enzyme supplementation may be practical tools to control enteric pathogens and benefit the intestinal health and food safety of the birds.

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