Short-chain fatty acids and insulin sensitivity: a systematic review and meta-analysis.

CONTEXT: There is substantial evidence that reduced short-chain fatty acids (SCFAs) in the gut are associated with obesity and type 2 diabetes, although findings from clinical interventions that can increase SCFAs are inconsistent. OBJECTIVE: This systematic review and meta-analysis aimed to assess the effect of SCFA interventions on fasting glucose, fasting insulin, and homeostatic model assessment of insulin resistance (HOMA-IR). DATA SOURCES: Relevant articles published up to July 28, 2022, were extracted from PubMed and Embase using the MeSH (Medical Subject Headings) terms of the defined keywords [(short-chain fatty acids) AND (obesity OR diabetes OR insulin sensitivity)] and their synonyms. Data analyses were performed independently by two researchers who used the Cochrane meta-analysis checklist and the PRISMA guidelines. DATA EXTRACTION: Clinical studies and trials that measured SCFAs and reported glucose homeostasis parameters were included in the analysis. Standardized mean differences (SMDs) with 95%CIs were calculated using a random-effects model in the data extraction tool Review Manager version 5.4 (RevMan 5.4). The risk-of-bias assessment was performed following the Cochrane checklist for randomized and crossover studies. DATA ANALYSIS: In total, 6040 nonduplicate studies were identified, 23 of which met the defined criteria, reported fasting insulin, fasting glucose, or HOMA-IR values, and reported change in SCFA concentrations post intervention. Meta-analyses of these studies indicated that fasting insulin concentrations were significantly reduced (overall effect: SMD = -0.15; 95%CI = -0.29 to -0.01, P = 0.04) in treatment groups, relative to placebo groups, at the end of the intervention. Studies with a confirmed increase in SCFAs at the end of intervention also had a significant effect on lowering fasting insulin (P = 0.008). Elevated levels of SCFAs, compared with baseline levels, were associated with beneficial effects on HOMA-IR (P < 0.00001). There was no significant change in fasting glucose concentrations. CONCLUSION: Increased postintervention levels of SCFAs are associated with lower fasting insulin concentrations, offering a beneficial effect on insulin sensitivity. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number CRD42021257248.

MicroRNA Profiling from Tears as a Potential Non-invasive Method for Early Detection of Diabetic Retinopathy.

Diabetic retinopathy (DR) is a vascular complication of diabetes that can lead to partial or complete loss of vision. Early detection and treatment of DR can prevent blindness. Regular clinical examination is recommended for DR diagnosis; however, it is not always possible or feasible due to limited resources, expertise, time, and infrastructure. Several clinical and molecular biomarkers are proposed for the prediction of DR including microRNAs. MicroRNAs are a class of small non-coding RNAs that are found in biofluids and can be measured using reliable and sensitive methods. The most commonly used biofluid for microRNA profiling is plasma or serum; however, tear fluid (tears) is also demonstrated to contain microRNAs. MicroRNAs isolated from tears present a non-invasive source for DR detection. Different methods of microRNA profiling are available including digital PCR-based methods that can detect up to a single copy of microRNA in the biofluids. Here, we describe microRNA isolation from tears using manual method as well as using a high-throughput automated platform followed by microRNA profiling using digital PCR system.

Short-chain fatty acid concentrations in the incidence and risk-stratification of colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: The beneficial role of gut microbiota and bacterial metabolites, including short-chain fatty acids (SCFAs), is well recognized, although the available literature around their role in colorectal cancer (CRC) has been inconsistent. METHODS: We performed a systematic review and meta-analysis to examine the associations of fecal SCFA concentrations to the incidence and risk of CRC. Data extraction through Medline, Embase, and Web of Science was carried out from database conception to June 29, 2022. Predefined inclusion/exclusion criteria led to the selection of 17 case-control and six cross-sectional studies for quality assessment and analyses. Studies were categorized for CRC risk or incidence, and RevMan 5.4 was used to perform the meta-analyses. Standardized mean differences (SMD) with 95% confidence intervals (CI) were calculated using a random-effects model. Studies lacking quantitation were included in qualitative analyses. RESULTS: Combined analysis of acetic, propionic, and butyric acid revealed significantly lower concentrations of these SCFAs in individuals with a high-risk of CRC (SMD = 2.02, 95% CI 0.31 to 3.74, P = 0.02). Additionally, CRC incidence was higher in individuals with lower levels of SCFAs (SMD = 0.45, 95% CI 0.19 to 0.72, P = 0.0009), compared to healthy individuals. Qualitative analyses identified 70.4% of studies reporting significantly lower concentrations of fecal acetic, propionic, butyric acid, or total SCFAs in those at higher risk of CRC, while 66.7% reported significantly lower concentrations of fecal acetic and butyric acid in CRC patients compared to healthy controls. CONCLUSIONS: Overall, lower fecal concentrations of the three major SCFAs are associated with higher risk of CRC and incidence of CRC.

Circulating microRNAs from early childhood and adolescence are associated with pre-diabetes at 18 years of age in women from the PMNS cohort.

With type 2 diabetes presenting at younger ages, there is a growing need to identify biomarkers of future glucose intolerance. A high (20%) prevalence of glucose intolerance at 18 years was seen in women from the Pune Maternal Nutrition Study (PMNS) birth cohort. We investigated the potential of circulating microRNAs in risk stratification for future pre-diabetes in these women. Here, we provide preliminary longitudinal analyses of circulating microRNAs in normal glucose tolerant (NGT@18y, N = 10) and glucose intolerant (N = 8) women (ADA criteria) at 6, 12 and 17 years of their age using discovery analysis (OpenArray™ platform). Machine-learning workflows involving Lasso with bootstrapping/leave-one-out cross-validation identified microRNAs associated with glucose intolerance at 18 years of age. Several microRNAs, including miR-212-3p, miR-30e-3p and miR-638, stratified glucose-intolerant women from NGT at childhood. Our results suggest that circulating microRNAs, longitudinally assessed over 17 years of life, are dynamic biomarkers associated with and predictive of pre-diabetes at 18 years of age. Validation of these findings in males and remaining participants from the PMNS birth cohort will provide a unique opportunity to study novel epigenetic mechanisms in the life-course progression of glucose intolerance and enhance current clinical risk prediction of pre-diabetes and progression to type 2 diabetes.

Analysis of Half a Billion Datapoints Across Ten Machine-Learning Algorithms Identifies Key Elements Associated With Insulin Transcription in Human Pancreatic Islet Cells.

Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow was tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1αß-/- mice that demonstrate dedifferentiation of pancreatic ß-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key elements associated with insulin transcription and provide workflows for future analyses.

A Pro-Endocrine Pancreatic Islet Transcriptional Program Established During Development Is Retained in Human Gallbladder Epithelial Cells.

BACKGROUND & AIMS: Pancreatic islet ß-cells are factories for insulin production; however, ectopic expression of insulin also is well recognized. The gallbladder is a next-door neighbor to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells. METHODS: We compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assays, RNA sequencing, real-time polymerase chain reaction, chromatin immunoprecipitation, and functional studies. RESULTS: We show that the epithelial lining of developing, as well as adult, mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a non-obese diabetic (NOD) mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Interestingly, in human gallbladders, insulin splice variants are absent, although insulin splice forms are observed in human islets. CONCLUSIONS: In summary, our biochemical, transcriptomic, and functional data in mouse and human gallbladder epithelial cells collectively show the evolutionary and developmental similarities between gallbladder and the pancreas that allow gallbladder epithelial cells to continue insulin production in adult life. Understanding the mechanisms regulating insulin transcription and translation in gallbladder epithelial cells would help guide future studies in type 1 diabetes therapy.

Long-term outcome of patients receiving haematopoietic allogeneic stem cell transplantation as first transplant for high-risk Hodgkin lymphoma: a retrospective analysis from the Lymphoma Working Party-EBMT.

We analysed long-term outcome of patients receiving haematopoietic allogeneic stem cell transplantation (allo-HSCT) as a first transplant for high-risk Hodgkin lymphoma (HL). One hundred and ninety patients were included in this study, 63% of them had previously received brentuximab vedotin and/or checkpoint inhibitors. Seventy patients (37%) received an unrelated donor allo-HSCT, 99 (51%) had myeloablative conditioning (MAC) and 60% had in vivo T-cell/depleted grafts (TCD). The 100-day cumulative incidence (CI) of grade II-IV acute graft-versus-host disease (GVHD) was 25% and the 3-year CI of chronic GVHD was 38%. The 3-year CI of non-relapse mortality (NRM) and relapse rate were 21% and 38% respectively. After a median follow-up of 58 months, 3-year overall survival (OS) and progression-free survival (PFS) were 58% and 41% respectively. Multivariate analysis showed that, in comparison to reduced-intensity conditioning regimens with or without TCD, MAC using TCD had similar NRM and a lower risk of relapse leading to significantly better OS and PFS. MAC without TCD was associated with higher NRM and worse survival outcomes. These results suggest that in patients with high-risk HL and candidates of allo-HSCT, a MAC strategy with TCD might be the best option.

Manipulating cellular microRNAs and analyzing high-dimensional gene expression data using machine learning workflows.

MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a).

Insulin micro-secretion in Type 1 diabetes and related microRNA profiles.

The aim of this cross-sectional study was to compare plasma C-peptide presence and levels in people without diabetes (CON) and with Type 1 diabetes and relate C-peptide status to clinical factors. In a subset we evaluated 50 microRNAs (miRs) previously implicated in beta-cell death and associations with clinical status and C-peptide levels. Diabetes age of onset was stratified as adult (≥ 18 y.o) or childhood (< 18 y.o.), and diabetes duration was stratified as ≤ 10 years, 10-20 years and > 20 years. Plasma C-peptide was measured by ultrasensitive ELISA. Plasma miRs were quantified using TaqMan probe-primer mix on an OpenArray platform. C-peptide was detectable in 55.3% of (n = 349) people with diabetes, including 64.1% of adults and 34.0% of youth with diabetes, p < 0.0001 and in all (n = 253) participants without diabetes (CON). C-peptide levels, when detectable, were lower in the individuals with diabetes than in the CON group [median lower quartile (LQ)-upper quartile (UQ)] 5.0 (2.6-28.7) versus 650.9 (401.2-732.4) pmol/L respectively, p < 0.0001 and lower in childhood versus adult-onset diabetes [median (LQ-UQ) 4.2 (2.6-12.2) pmol/L vs. 8.0 (2.3-80.5) pmol/L, p = 0.02, respectively]. In the childhood-onset group more people with longer diabetes duration (> 20 years) had detectable C-peptide (60%) than in those with shorter diabetes duration (39%, p for trend < 0.05). Nine miRs significantly correlated with detectable C-peptide levels in people with diabetes and 16 miRs correlated with C-peptide levels in CON. Our cross-sectional study results are supportive of (a) greater beta-cell function loss in younger onset Type 1 diabetes; (b) persistent insulin secretion in adult-onset diabetes and possibly regenerative secretion in childhood-onset long diabetes duration; and (c) relationships of C-peptide levels with circulating miRs. Confirmatory clinical studies and related basic science studies are merited.

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues.

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic ß-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription.

Urinary microRNAs as non-invasive biomarkers for toxic acute kidney injury in humans.

MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell's viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell's viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p < 0.0001) and receiver operator characteristics area-under-curve (ROC-AUC) > 0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers.

Postpartum circulating microRNA enhances prediction of future type 2 diabetes in women with previous gestational diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes mellitus is a major cause of morbidity and death worldwide. Women with gestational diabetes mellitus (GDM) have greater than a sevenfold higher risk of developing type 2 diabetes in later life. Accurate methods for postpartum type 2 diabetes risk stratification are lacking. Circulating microRNAs (miRNAs) are well recognised as biomarkers/mediators of metabolic disease. We aimed to determine whether postpartum circulating miRNAs can predict the development of type 2 diabetes in women with previous GDM. METHODS: In an observational study, plasma samples were collected at 12 weeks postpartum from 103 women following GDM pregnancy. Utilising a discovery approach, we measured 754 miRNAs in plasma from type 2 diabetes non-progressors (n = 11) and type 2 diabetes progressors (n = 10) using TaqMan-based real-time PCR on an OpenArray platform. Machine learning algorithms involving penalised logistic regression followed by bootstrapping were implemented. RESULTS: Fifteen miRNAs were selected based on their importance in discriminating type 2 diabetes progressors from non-progressors in our discovery cohort. The levels of miRNA miR-369-3p remained significantly different (p < 0.05) between progressors and non-progressors in the validation sample set (n = 82; 71 non-progressors, 11 progressors) after adjusting for age and correcting for multiple comparisons. In a clinical model of prediction of type 2 diabetes that included six traditional risk factors (age, BMI, pregnancy fasting glucose, postpartum fasting glucose, cholesterol and triacylglycerols), the addition of the circulating miR-369-3p measured at 12 weeks postpartum improved the prediction of future type 2 diabetes from traditional AUC 0.83 (95% CI 0.68, 0.97) to an AUC 0.92 (95% CI 0.84, 1.00). CONCLUSIONS: This is the first demonstration of miRNA-based type 2 diabetes prediction in women with previous GDM. Improved prediction will facilitate early lifestyle/drug intervention for type 2 diabetes prevention.

Promoting Pro-Endocrine Differentiation and Graft Maturation Following Surgical Resection of the Mouse Pancreas.

Type 1 diabetes (T1D) is an autoimmune disease, where insulin-producing ß-cells in the pancreas are inappropriately recognized and destroyed by immune cells. Islet transplantation is the most successful cell-based therapy for T1D individuals who experience frequent and severe life-threatening hypoglycemia. However, this therapy is extremely restricted owing to the limited availability of donor pancreas. In recent years, significant progress has been made in generating ß-cells from stem/progenitor cells using different approaches of in vitro differentiation. The insulin production from such in vitro generated ß-cells is still far less than that observed in islet ß-cells. We employed a novel strategy to improve the efficiency of progenitor cell differentiation by performing partial mouse pancreas resection after transplanting in vitro generated insulin-producing cells under the kidney capsule of these mice. Pancreas resection (pancreatectomy) has been shown to induce regenerative pathways, leading to regeneration of almost the entire resected pancreas over 3-5 weeks in mice. We found that in our method, regenerating mouse pancreas promotes better graft differentiation/maturation and insulin production from transplanted cells. In this chapter, we detail the protocols used for transplantation of in vitro differentiated cells in immunocompromised mice, partial pancreatectomy in host (NOD scid) mice, and assessment of graft function. We believe that our protocols provide a solid platform for further studies aimed at understanding growth/differentiation molecules secreted from regenerating pancreas that promote graft maturation.

Directed differentiation into insulin-producing cells using microRNA manipulation.

Our commentary is focused on three studies that used microRNA overexpression methods for directed differentiation of stem cells into insulin-producing cells. Islet transplantation is the only cell-based therapy used to treat type 1 diabetes mellitus. However, due to the scarcity of cadaveric donors and limited availability of good quality and quantity of islets for transplant, alternate sources of insulin-producing cells are being studied and used by researchers. This commentary provides an overview of distinct studies focused on manipulating microRNA expression to optimize differentiation of embryonic stem cells or induced pluripotent stem cells into insulin-producing cells. These studies have used different approaches to overexpress microRNAs that are highly abundant in human islets (such as miR-375 and miR-7) in their differentiation protocol to achieve better differentiation into functional islet beta (ß)-cells.

An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length.

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease.

A Proteomic View of the Cross-Talk Between Early Intestinal Microbiota and Poultry Immune System.

Proteomics has been used to investigate cross-talk between the intestinal microbiome and host biological processes. In this study, an in ovo technique and a proteomics approach was used to address how early bacterial colonization in the gastrointestinal tract (GIT) could modulate inflammatory and immune responses in young broilers. Embryos at 18 embryogenic days were inoculated with saline (S), 102 CFU of Citrobacter freundii (CF), Citrobacter species (C2), or lactic acid bacteria mixture (L) into the amnion. At 10 days posthatch, ileum samples from 12 birds per treatment were selected for tandem mass spectrometry analysis. Our further findings indicated that treatment-specific influences on early GIT microbiota resulted in different immune responses in mature broilers. Predicted functional analyses revealed activation of inflammation pathways in broilers treated in ovo with L and CF. Exposure to L enhanced functional annotation related to activation, trafficking of immune cells, and skeletal growth based-network, while CF inhibited biological functions associated with immune cell migration and inflammatory response. These results highlighted that proper immune function was dependent on specific GIT microbiota profiles, in which early-life exposure to L-based probiotic may have modulated the immune functions, whereas neonatal colonization of Enterobacteriaceae strains may have led to immune dysregulation associated with chronic inflammation.

Impact of in ovo administered pioneer colonizers on intestinal proteome on day of hatch.

Pioneer colonization of the gastrointestinal tract (GIT) by bacteria is thought to have major influence on neonatal tissue development. Previous studies have shown in ovo inoculation of embryos with saline (S), species of Citrobacter (C, C2), or lactic acid bacteria (L) resulted in an altered microbiome on day of the hatch (DOH). The present study investigated GIT proteomic changes at DOH in relation to different inoculations. Embryos were inoculated in ovo with S or ∼102 cfu of C, C2, or L at 18 embryonic days. On DOH, the GIT was collected, and tissue proteins were extracted for analysis via tandem mass spectrometry. A total of 493 proteins were identified for differential comparison with S at P ≤ 0.10. Different levels were noted in 107, 39, and 78 proteins in C, C2, and L groups, respectively, which were uploaded to Ingenuity Pathway Analysis to determine canonical pathways and biological functions related to these changes. Three members of the cytokine family (interleukin [IL]-1ß, IL6, and Oncostatin M) were predicted to be activated in C2, indicated with Z-score ≥ 1.50, which suggested an overall proinflammatory GIT condition. This was consistent with the activation of the acute-phase response signaling pathway seen exclusively in C2 (Z-score = 2.00, P < 0.01). However, activation (Z-score = 2.00) of IL-13, upregulation of peroxiredoxin-1 and superoxide dismutase 1, in addition to activation of nitric oxide signaling in the cardiovascular system of the L treatment may predict a state of increased antioxidant capacity and decreased inflammatory status. The nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response (Z-score = 2.00, P < 0.01) was predicted to be upregulated in C which suggested that chicks were in an inflammatory state and associated oxidative stress, but the impact of these pathways differed from that of C2. These changes in the proteome suggest that pioneer colonizing microbiota may have a strong impact on pathways associated with GIT immune and cellular development.

Research Note: Evaluating fecal shedding of oocysts in relation to body weight gain and lesion scores during Eimeria infection.

Coccidiosis has been a pervasive disease within the poultry industry, with test parameters used to measure effectiveness of treatment strategies often being subjective or influenced by non-disease-related activity. Four experiments were completed, which examined several test parameters of coccidiosis, including body weight gain (BWG), lesion scores, and oocysts per gram of feces (OPG). Each experiment included at least 2 parameters for measuring coccidial infection in chickens and turkeys. In experiment 1, an inoculated control was measured against 3 anticoccidial groups, whereas in experiments 2 to 4, noninoculated and inoculated controls were compared via BWG and OPG. Lesion scores were also included in experiments 1, 3, and 4. Experiment 4 resulted in high correlation, via Pearson correlation coefficient, between BWG and OPG (r = -0.69), very high correlation between OPG and lesion score (r = 0.86), and moderate correlation between BWG and lesion score (r = -0.49). Lesion scores proved to be effective in confirming Eimeria infection, although they did not correlate well with BWG or OPG. Each parameter tended to provide more useful information when lined up with the Eimeria life cycle. Incorporation of OPG, with BWG and lesion scores, as test parameters to measure coccidiosis intervention strategies, provides a global description of disease that may not otherwise be observed with the 2 latter measurements alone.

Preliminary studies on development of a novel subunit vaccine targeting Clostridium perfringens mucolytic enzymes for the control of necrotic enteritis in broilers.

Necrotic enteritis (NE) is a pervasive enteric disease responsible for large scale economic losses within the global poultry industry. The etiologic agent of NE is Clostridium perfringens (CP), an opportunistic pathogen that utilizes numerous extracellular toxins and glycoside hydrolases (GH) as key virulence and nutrient acquisition factors. Notably, some GH, mucinases, degrade components of mucin in the gastrointestinal tract as an energy source. Targeting this mechanism may serve to reduce the incidence of disease associated with CP. Two experiments were completed that evaluated mucinase vaccine targets sourced from conserved peptide sequences of carbohydrate binding module 32 of CP mucinases. In experiment 1, 37 antigen peptides were synthetically generated and used to produce hyper-immune sera, which was then evaluated for ability to obstruct CP growth in vitro. Total CFU of CP were measured at 4, 6, and 8 h incubation to determine growth rate. Peptides 4, 5, 22, 24, and 30 were selected for further in vivo testing based on conservation or the ability to inhibit CP growth by over 50% at 6 and 8 h. In experiment 2, the aforementioned peptides were conjugated to an agonistic, CD40-targetting antibody and evaluated in vivo. Broilers were given an Eimeria maxima and CP in order to induce NE and assess vaccine efficacy. Treatments included a non-vaccinated non-inoculated control, non-vaccinated inoculated control (NVIC), vaccination with peptide 4, 5, 22, 24, or 30 (VP4-VP30), or a combination of all 5 peptides (MC). There was a significant increase (P < 0.05) in the percent change in BWG relative to NVIC for vaccination with peptide 22 and MC of 18.54 and 17.43%, respectively. MC vaccinated group had the lowest lesions with a mean score of 0.63 ± 0.18. These results suggest the MC combination was the most successful in alleviating overall performance losses associated with NE-infected broilers and encourage future testing of MC in the development of an NE vaccine.