Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Moringa oleifera is an important food commodity that is a nutritionally rich source of proteins, vitamins and minerals, as well as a variety of phytochemicals that are important for health/immunity and mitigation of disease. Moringa is not native to North Carolina. The purpose of this project is to establsish M. oleifera plant in North Carolina the most potent Moringa cultivars with the highest bioactive compounds at the NCA&T University farm and explore the potential of M. oleifera as a supplemental food source for animals. As part of this project several experiments have been conducted that investigated it as a potential treatment for infection, evaluated its role in inflammation and examined its potential utility in promoting growth and health. The outputs of this year of the project are described. Experiments have been fruitful and data generated from them have been presented by poster at several conferences. We were able to yield 200 lbs/dry weight from a 2.5 acre plot of Moringa plated on the farm and protocols for cultivation of Moringa trees and harvesting/drying of leaves. Students and Faculty attended the NC- Louis Stokes Alliances for Minority Participation 2012 annual research conference on September 21, 2012 and the North Carolina Alliance to Create Opportunity through Education Alliance Day 2012 "Empowering the Next Generation of Leaders" October 16, 2012. Two seminars presenting results of the effect of Moringa on inflammation using a mouse model have been delivered and a poster presentation at the Society of Leukocyte biology meeting was made. Furthermore, three abstracts, each for oral presentation have been submitted and accepted for the Association of Research Directors (ARD) 2013 meeting. Abstracts will be submitted to the 2013 JAM of Animal & Dairy Sciences, Indianapolis, IN. and manuscripts are in preparation for submission in 2013. Experimentation is ongoing. PARTICIPANTS: In total, there have been 9 faculty members involved in this project. The faculty have come from within the departments of animal sciences (4), ag extension/forestry (2), family consumer sciences (1), chemistry (1), and biology. Because of the collaborations with faculty from the Chemistry and Biology Departments two undergraduate students have been involved. Professional development of students and PI was afforded because of attendance to conferences. The opportunities for training include; 1) participation of a high school student accepted into the Research Apprenticeship Program of the School of Environmental Sciences and NCA&T. In addition, four undergraduate students, five-master's level graduate students, and one postdoctoral fellow have participated on this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A summary of experiments are presented. 1) Establishment of quality Moringa seedlings in the greenhouse was conducted. High quality seedlings were identified sorted for planting in the high tunnel and field. At the NCA&T farm researchers intercropped pecan and Moringa using a 2.5 acres alley cropping system. Plants developed new shoots and branches before harvest. Harvested Moringa leaves were dried and stored in plastic bags. Extracts were prepared and compared with the extracts from leaves of trees grown in Ghana and Winston-Salem, NC. 2) Mice were given water or Moringa tea (MT) for seven days and then challenged intranasally with either saline, Lipopolysaccaride (LPS) or extracts from swine facility dust (DE)to assay the anti-inflammatory properties. Mice that consumed MT and challenged with LPS or DE had significant decreases in the inflammatory responses of the upper respiratory tract after challenge suggesting that Moringa leaves have anti-inflammatory properties. 3) The effect of Moringa on antioxidant enzymes in plasma, white blood cell (WBC) counts, cell viability, and inflammatory cytokine/gene expression by neutrophils isolated from cows, sheep and goats was examined. Moringa improved cell viability in sheep and goats. Species-specific effects were observed for other interleukins. Moringa increased the percentage of lymphocytes and decreased neutrophils in WBC. Real time PCR of isolated neutrophils showed that supplementation by Moringa resulted in up regulation of 5 inflammatory genes in cows, 26 in goats, and 25 in sheep. In contrast, 1 gene in cows and 25 in sheep were down regulated. The effects of Moringa leaf-aqueous extracts on Boer goats infected with gastrointestinal parasites were evaluated. Moringa treatment did not affect Coccidia egg counts, FAMACHA score, PCV or body weight. Moringa treatment increased mononuclear cells and decreased Haemonchus eggs. We conclude from this study that Moringa supplementation modulates inflammatory gene expression, oxidative stress and parasite burden in a species-specific manner through ruminant cell mediated immunity. Preliminary results from another independent small study in goats suggest that Moringa consumption had a positive effect on animals with low worm counts but there was no effect on animals with high-count Haemonchus infection. 4) A study to evaluate the chemical composition of the leaf biomass and its nutritional value to nursery pigs was done. Three groups of pigs (5/group) were fed 3 types of diets with/without moringa leaf meal (MLM) and effects on average daily feed intake was calculated, and the general health and wellbeing of nursery-age pigs was monitored. Analyzed MLM were comparable to a typical plant fodder used to feed animals concerning levels of dry matter, organic matter, protein, and aNDF. Further analysis of leaves showed them to contain an appreciable amount of nutrients but further studies of their availability to the animal is needed. No clear advantages of feeding MLM on feed intake and performance of pigs was observed. Collected Fecal swab samples are being analyzed to identify/enumerate bacteria associated with the gut health.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Moringa oleifera is an important food commodity that is a nutritionally rich source of proteins, vitamins and minerals, as well as a source for a variety of phytochemicals that are important for health, immunity and mitigation of disease. The purpose of this project is to explore the potential of M. oleifera as a: (a) treatment source for infection and inflammation and (b) supplemental food source for promoting growth and health of animals. The project aims are; (1) Investigate the function of B and T lymphocytes that are exposed to Moringa in vivo using a mouse model in a study of immune modulation study. For aim 1, a preliminary study was conducted with three objectives to allow for optimization of the mouse model. Objective 1: devise a method for preparation of a Moringa tea., Objective 2: compare water and Moringa tea consumption by mice and Objective 3: Assess white blood cell count in the blood in mice in the study. Aim (2): Investigate the effect of Moringa supplementation on performance of growing pigs (pig growth study), and Aim (3): Investigate the effect of Moringa supplementation for treatment potential for controlling mastitis in ruminants (cow mastitis study). For completion of Aim 3, there are two objectives. Objective 1: Elucidate in vitro effects of Moringa extracts on gene expression in three types of ruminants: cows, sheep, and goats and Objective 2: Evaluate the natural response to Moringa supplementation in dairy cows. From the initiation of this project, the outputs have been as follows. 1) IACUC approval was obtained to conduct in vitro and in vivo studies. 2) Students, undergraduate and graduate (MS) have been hired. 3) Two small shipments of dried Moringa leaves (DML) from a provider in Winston Salem, NC have been received. 4) A process for logging and tracking Moringa samples for chemical and active compounds has been devised. 5) Chemical and active compound analysis of the DML has begun. 6) Experiments toward aims 1 and 3 have begun and are presented. For aim 1, Moringa tea or water was administered to six mice and blood cell levels assessed. For Aim 3, sun dried Moringa leaves were pulverized to a fine homogeneous powder and used to prepare an extract using the Soxhlet method. Extracts were used to treat whole blood and isolated neutrophils from cows, sheep and goats (3 each). The effect of Moringa exposure on white blood cell differential counts, isolated neutrophil viability, RNA purity and concentration and secretion of cytokines was examined. Further, the imunomodulatory role in exposure to bacterial lipopolysacharide or peptidoglycan was evaluated in isolated neutrophils. Preliminary statistical analyses have been completed using SAS. All Moringa extracts have been sent to N.C. A&T's Center for Excellence in Post Harvest Technologies at the North Carolina Research Campus for chemical analysis. PARTICIPANTS: The PI an assistant Professor of Immunology, and co-PIs an associate professor of animal sciences and animal nutrition and a Professor of animal sciences and biotechnologist, all from the Department of Animal Sciences are each involved in investigating the effect of Moringa on Animal Health. An additional co-PI an Associate Professor Department of Family and Consumer Sciences and interim director of the Center for Excellence in Post-Harvest Technologies, has been leading the chemical and active compound analysis of Moringa. The opportunities for training include; 1) participation of a high school student accepted into the Research Apprenticeship Program of the School of Environmental Sciences and NCAT. During the 4-week program, the HS student worked in the laboratory the PI and established a protocol for feeding Moringa to mice, and evaluated white blood cell counts of mice that consumed it. In addition, one undergraduate student and two-master's level graduate students as well as one postdoctoral fellow in the Department of Animal Sciences have participated on this project. Currently, no additional research staff is being supported on this project. However, we anticipate hiring a part-time graduate student or undergraduate apprenticeship student to assist in the preparation and analysis of antimicrobial properties of dried leaves, solvent and water crude extracts. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Due to difficulty in acquiring large enough quantities of Dried Moringa leaves to feed Dairy Cattle the following changes in the project approach will occur. Moringa Leaves obtained from the Winston Salem provider will be used to prepare extracts for evaluation of markers of innate immunity relevant to infection in goats at the NC A&T herd. The leaves have been processed to powder form and the study will commence after IACUC approval.

Impacts
The findings and results of the activities that have occurred to date are still preliminary, and therefore have not resulted in significant outcomes/impacts. Although there are no significant changes in knowledge that have occurred to date following are are the results of the activities to date: In vivo analysis of Moringa consumption on immune modulation using a mouse model shows that feed and fluid consumption of the mice in the study were similar. Blood analysis show that mice that consumed Moringa tea for 7 days had significantly greater levels (p=0.0091 by unpaired two tailed student t- test) of white blood cells, as compared to control mice that consumed water. Further analysis show that there was a significant increase (p=0.0069 by unpaired two tailed student T test) in the lymphocyte population and a slight increase in the monocyte population of the Moringa tea group as compared to the controls. In vitro analysis indicates that treatment of whole blood and isolated neutrophils from cows, sheep and goats with Moringa extract resulted in increased mononuclear cell percentages and decreased viability of isolated neutrophils (p<0.05). Animal variation and species-specific effects of Moringa were observed. Sufficient RNA has been isolated for evaluation of the impact on gene transcription. In addition, differential effects were observed on the secretion of eight cytokines tested. Preliminary results indicate that Moringa extract has differential effects that affect ruminant immunity in vitro (in lab tests). The funding and resources from USDA Evans-Allen has enabled the Center for Excellence in Post Harvest Technologies to develop a robust sampling procedure to extract and quantify the different levels of phytonutrients in dried Moringa leaves. The nutritional (proximate) analysis of dried plant leaves of Moringa was determined. Results showed that the leaves were rich in potassium (2000 mg/100g); total Vitamin A (10,400 IU/100 g); Vitamin B-carotene; Iron (63.9 mg/100g), and Folate/folic acid (1061 mcg/100g) using standard AOAC modified methods. Analysis of Moringa extracts and tea are forth coming. Finally, the resources leveraged by this project have been utilized to pay student salaries, purchase supplies, reagents and Moringa leaves, and therefore have been and are essential to the progress and success of this project. A bulk purchase of Moringa leaves has been made and progress in Aim 2, to Investigate the effect of Moringa supplementation on performance of growing pigs, will be achieved in year two of the funding.

Publications

• No publications reported this period