Recipient Organization
DELAWARE STATE UNIVERSITY
1200 NORTH DUPONT HIGHWAY
DOVER,DE 19901
Performing Department
Agriculture & Natural Resources
Non Technical Summary
The small ruminant enterprise lends itself to small and beginning farmers wanting to be involved in livestock production. However,small ruminants are affected significantly by gastrointestinal n ematodes (GINs), most notably the parasiteHaemonchus contortus,which causes significant economic losses inproduction.In the absence of vaccines,GINs are controlled using broad-spectrum anthelmint ic drugs.However, relying heavily on the available anthelmintic (dewormer) treatments for parasite controlhas led to the emergence of multi-drug resistant (MDR) parasites which pose a significant challenge to parasite control on small ruminant farms world-wide.Research conducted in the United States confirm ahigh prevalence offarms with MDR and even resistance to all classes of available anthelmintics.Hence, there is an urgent need to identify alternatives to anthelmintics that can be used in small ruminants to reduce problems associated with GIN infection.One mechanism ofidenti fying an effective natural (plant) product that can serve as a dewormer is by conducting whole-organism high throughput screening (HTS), which screens a multitude of possible dewormers to identif y those showing anthelmintic properties against the different life stages of the parasite. This project aims to (1)screen extracts fromCannabis sativa(hemp),Sambucus nigraL.ssp. (elderberry), and Cucurbita pepo(pumpkin)to identify theirin vitroanthelmintic effects againstHaemonchus contortusand other gastrointestinal nematodes; and (2) test the cytotoxicity of these plant extracts on mamm alian white blood cells. Results from this project will be used to promote further testing of plants with anthelmintic properties in small ruminants and will be disseminated to animal scientists, extension agents, and producers through presentations at scientific meetings, producer workshops, newsletters, and journal publications. It will provide students with experiential learning oppor tunities, which should help them consider future careers in small ruminants and animal sciences research and extension programs.
Animal Health Component
12%
Research Effort Categories
Basic
75%
Applied
12%
Developmental
13%
Goals / Objectives
(N/A)
Project Methods
Parasites for these experiments will be obtained from Savanna crossbred goats housed on DSU Hickory Hill farm who are naturally infected on pasture or artificially infected withH. contortus. The naturally infected animals will be used mainly for adultH. contortuscollection or fecal sample collection. The fecal samples will be used if the chemical will be tested on a conglomerate of thir d stage larval parasites. On the other hand, goats inoculated with parasites will be used only to acquire fecal samples for culturing ofH. contortusL3 and L4 parasites. Fecal samples will be coll ected rectally from each animal and used to conduct fecal egg count (FEC) via the modified McMaster technique to determine the number of probableH. contortuseggs per gram (epg) of feces present. When FEC are greater than (gt;) 850 epg, the infected animals will be used to conduct the experiments below. To ensure that the parasites we are using for thein vitroanalysis of chemicals with anthelmintic properties areH. contortusand to also have a constant supply of these parasites, it is necessary that we inoculate goats with this parasite.To acquire infective third stageH. contort uslarvae, two wethers (castrated male goats) 12 ndash; 18 months old will be used for the inoculation and parasite generation protocol. The goats used in this protocol will be dewormed with moxid ectin (0.4 mg/kg), valbazen (20 mg/kg) and levamisole (12 mg/kg) on two consecutive days in order to kill all parasites. Fecal egg count will be performed at 7, 14, and 21 days to monitor parasit e reduction. Following a 21-day dewormer withdrawal period, goats will be artificially inoculated orally with5,000 third-stage drug-susceptible larvae (L3) and held in 2.2 meters x 4.4 meters pen with straw bedding to continuously provideH. contortuseggs to generate larvae. Animals will be fed a 15% crude protein ration at 3% of their body weight and have access to water and hayad libitu m. They will be observed daily and fecal and blood samples will be taken bi-weekly to monitor fecal egg counts and packed cell volume (PCV).The PCV is a system used for testing anemia levels of a n animal, by calculating the ratio of red blood cells to the whole blood collected in capillary tubes before centrifugation. If the PCV is lt;16%, animals will be dewormed with all three classes of dewormer (moxidectin (0.4 mg/kg), valbazen (20 mg/kg) and levamisole (12 mg/kg)) and removed from the pen in order to shed/release the parasites. Every year throughout this study, two differen t wethers will be selected, and the other wethers will be treated and removed from the pen. The wethers that were treated will be allowed to go through deworming withdrawal (to ensure no contamin ants in the meat) and then sold when all other culled animals are sold. Wethers will be selected because they can fight off the parasite infection on their own before it gets lethal. Plant extr acts needed for this project will be isolated fromCannabis sativa(hemp),Sambucus nigraL.ssp. (elderberry), andCucurbita pepo(pumpkin)per the method developed in a lab at DSU. In brief, plants wil l behomogenized with one of four solvents: acetone, methanol, ethanol, and water. The solvent in the decanted filtrate will be removed and extract dried using a rotary evaporator. The dried extra cts will be stored at 4oC until needed. Haemonchus contortusL3 parasites will be harvested from fecal cultures according to amodified version of the guidelines put forth by Zajac and Conboy, 20 12.Briefly, a minimum of 10 grams of pooled fecal samples (from at least 10 goats heavily infected withGIN; based on fecal egg counts in combination with FAMACHAcopy; scores indicative of anemia) will be collected and placed in 500 ml beakers with vermiculite added at roughly a 1:1 ratio with feces to maintain moisture. Water (~5 ml) will be used to dilute feces and promote mixing with t he vermiculite. Each jar will then be labeled with a collection/set date and harvesting date and incubated for seven days at 27deg;C.After seven days samples will be Baermannized in warm (27oC) a utoclaved,distilled water (dH2O) will be poured into a funnel with frac14; inch wire screening in the bottom, and a tube with a clamp attached to the end for 12 ndash; 24 hours.Thereafter, larvae will be collected from the Baermann apparatus into two 50 ml conical tubes and then refrigerated for three hours to allow larvae to settle. The supernatant in both tubes will be aspirated and th e larvae combined into one tube and washed with sterile phosphate buffer saline (PBS; pH 7.4) ordH2O. After, larvae will be quantified andstored at a concentration of 150,000/25 ml of dH2O/PBS in T25 culture flask at 4oC for further use. Fourth-stage H. contortus larvae will be generated according to the guidelines from the University of Georgiarsquo;s Department of Infectious Diseases . Briefly, the sheaths from third-stage H. contortus larvae (L3) will be removed by placing 250,000 L3 larvae into a 15 ml centrifuge tube with 10 ml of 6% Clorox (sodium hypochlorite) and then s haken for 3-5 minutes at room temperature. The parasites will then be checked by microscopy to ensure the protocol was at least 90% successful. Once the exsheathment process is satisfactory (gt;9 0%), larvae will be washed with PBS solution using centrifugation at 1500 rpm for 5 minutes. The supernatant will be removed and the washing process repeated at least two additional times to remo ve all traces of the Clorox bleach. After which, parasites will be used to test extracts in the high throughput screening process in 24 well plates. Adult femaleH. contortusworms will be collec ted from naturally infected goats slaughtered in demonstration workshops for the mobile meat processing unit(developed through a previously funded 1890 Capacity Building Grant)or already destined for market/harvest from DSUrsquo;s goat herd (15 to 20 goats) at a USDA inspected abattoir.After harvest, the abomasum will be immediately excised and live femaleH.contortusadult worms will be i dentified (by the presence of white ovaries and red intestines due to their blood sucking ability) and isolated from the abomasal contents using spatula or forceps and placed in sterile PBS solut ion and maintained at 37deg;C.The parasites will be washed in sterilePBS followed by RPMI medium (pH 6.8, supplemented with 2% glucose, 500 units/ml penicillin, 500mu;g/ml streptomycin, 1.25mu;g/ ml amphotericin and 1mM chloramphenicol; Rhoads and Fetterer, 1995) and then used in extract screening. For extract screening,H. contortuslarvae and adult parasites will be cultured in RPMI-164 0 medium (pH = 6.8) supplemented with 2% glucose, 500 units/ml penicillin G, 500micro;g/ml streptomycin, 1.25 micro;g/ml amphotericin B and 1 mM chloramphenicol. Parasites will be allocated into 24 well plates (third-stage or fourth-stage larvae at 100/well) or six well plates (adults at 5/well) in triplicates with varying concentrations of the plant extractsand incubated at 37deg;C with 5% CO2for up to 48 hours. Additionally, control cultures without a plant extract will be set up with and without equivalent volumes of solvent.After 12, 24, 36, and 48 hours of culture, parasite motility of worms and larvae in each well will be tracked and quantified using thecomplete turnkey WormLab tracking solution and microscopy. The IC50 of the extracts will then be determined base d on the percentage of dead parasites at each extract concentration tested using nonlinear regression analysis by Graph Pad Prism software program. After screening of plant extracts, RNA will be extracted from parasites in each well or petri dish and stored at -80 deg;C until further use. After identifying plant extracts with anthelmintic effects on larvae and/or adultH. contortus, cyt otoxicity assays will be carried out by Dr. Matthews, seasonal technician, and graduate and undergraduate students to identify the IC50 for each extract on mammalian cells.Briefly, the cytotoxic concentrations of each plant extract with anthelmintic effects will be determined in mammalian cells using a colorimetric assay with the cell proliferation reagent WST-1 (Roche) for the quantific ation of cell viability on MonoMac cells (Ziegler-Heitbrock et al., 1988). Peripheral blood mononuclear (PBMC) will be isolated from goats and used in this proposed experiment. Cells will be seed ed at 104/well in RPMI-1640 medium (No phenol red) supplemented with 10% fetal calf serum, 1x nonessential amino acids, OPI medium (Hybri Max), and 1% penicillin-streptomycin in 96 well plates. T reatment will include plant extracts at increasing concentrations, controls without plant extract but equivalent volumes of solvent, and controls without extracts and solvent all in triplicate we lls. Cells will be cultured for 48 hours at 37deg;C with5% CO2andthen another one hour after the addition of 20mu;l of the WST-1 reagent to each well. One hundred microliter of medium from each w ell will be added to a new 96-well plate and quantified using a scanning multi-well spectrophotometer. Throughout the project period, extension programing will be conducted to address the needs of Delawarersquo;s small ruminant producers with respect to internal parasite management.