Final Report for GNE14-071
Gastrointestinal nematode (GIN) infections are a major health concern for small ruminant producers in the Northeastern United States. Anthelmintic resistance can be seen in all species of GIN. In search for alternative methods of GIN control in small ruminants, one of the most promising findings has been the discovery that some forages containing condensed tannins, also called proanthocyanidins (PAC), suppress GIN infection. Cranberries contain high levels of PAC. The objective of this study was to determine the anthelmintic efficacy of cranberry vine (CV) against an experimental infection of Haemonchus contortus in lambs. Twenty-one lambs were experimentally infected with 10,000 infective H. contortus L3 larvae. After the infection matured, lambs were assigned to receive one of three treatments, control, fed 200 grams of chopped alfalfa hay (Control, n=7), or 100 (CV100, n=7) or 200 grams (CV200, n=7) of dried and chopped CV for five weeks. Pruned cranberry vines (‘Stevens’ cultivar) were obtained in the spring of 2015 as the cranberry vines were coming out of dormancy. Weekly fecal egg counts (FEC) and packed cell volume (PCV) was determined. At the conclusion of the feeding trial, lambs were sent to the abattoir and their abomasa were opened for quantification of individual lambs’ worm burden and collection of adult worms for scanning and transmission electron microscopy. An increase in FEC over the feeding period was observed in the Control and CV100 lambs that did not occur in the CV200 lambs. Furthermore, there was an increase in PCV over time only in the CV200 lambs. Observations made using scanning and transmission electron microscopy indicate potential effects on the cuticle and feeding ability of the CV200 worms. Detailed results will be provided after the publication of this data.
The purpose of this project was to determine the anthelmintic efficacy of cranberry vine, at varying concentrations, against experimental Haemonchus contortus infection in lambs. Gastrointestinal nematode (GIN) parasite infections are a major health concern for small ruminant producers. GIN infections can be harmful to sheep and goats causing anemia, poor body condition, diarrhea and death; this results in economic losses for sheep and goat producers. The nematodes that account for the majority of economic and production losses are the barber pole worm (Haemonchus contortus), the brown stomach worm (Teladorsagia circumcincta) and the bankrupt worm (Trichostrongylus columbriformis). Haemonchus contortus is the most pathogenic GIN and responsible for the majority of production losses. The barber pole worm feeds on the blood of its host, leading to anemia. A heavy worm burden in an animal can result in the loss of about ½ cup of blood each day and ultimately be fatal. This problem is usually seen in young animals and periparturient females, which are more susceptible to H. contortus infections.
With the growth of anthelmintic resistance in GIN due to the overuse of commercially available anthelmintics (dewormers), alternative methods are needed for GIN control. An anthelmintic effect on GIN was demonstrated in sheep and goats consuming condensed tannin containing forages. One of the most heavily researched condensed tannin forages effective in managing GIN infections is lespedeza cuneata (Sericea lespideza), however it is not a suitable for the New England climate.
This research is focused on finding viable alternatives to commercial drugs, specifically examining the use of condensed tannin containing plants. Cranberries contain high levels of condensed tannins, also referred to as proanthocyanidins (PAC) and are grown in New England. Cranberry vines are routinely pruned as they are coming out of winter dormancy and as such may hold potential as an affordable option for producers for control of GIN.
In this study, we investigated the use of dried, chopped cranberry vines (CV) against GIN infections of H. contortus. Our investigation of CV is the first study that is looking at cranberry vine PAC as a natural alternative to commercial dewormers. Previous in vitro data in our laboratory with CV found an anthelmintic effect of an aqueous extract of ground CV as well as an organic extract of ground CV against H. contortus eggs, larvae and adult worms. An in vivo trial using three consecutive 26 gram oral doses of CV suspended in water to lambs experimentally infected with H. contortus was also conducted. This in vivo study demonstrated a slight reduction in fecal egg count in lambs two weeks after being drenched with CV for three consecutive days. Building on our in vitro and in vivo preliminary data, the next step of the whole animal trials was to look at the anthelmintic efficacy of CV at greater concentrations for a longer treatment period. Additionally, we needed to find an alternate way to administer the CV to the lambs; providing the CV to the lambs in the form of a pelleted supplement would meet this need. Previous work with sericea lespedeza, a condensed tannin containing forage, successfully utilized a pellet to administer the forage to the small ruminants.
The specific objectives of this study are:
- To develop a grain supplement containing varying concentrations of cranberry vine that lambs will readily consume.
- To determine the anti-parasitic potential of varying concentrations of cranberry vine provided daily, through a grain supplement, to lambs experimentally infected with Haemonchus contortus.
Objective 1: Difficulty obtaining access to a grinder that would process the CV fine enough for pelleting in the quantities needed, coupled with the discovery that lambs readily ate the CV when mixed with a chopped alfalfa hay and molasses, led to the decision to feed the chopped CV directly to the lambs instead of incorporating it into a pellet.
Objective 2: This objective was accomplished. Lambs, experimentally infected with Haemonchus contortus, were fed 0, 100, or 200 grams of CV daily for five weeks and fecal egg count and packed cell volume were monitored weekly. At the conclusion of the trial, the abomasum of each lamb was collected and individual worm burden was determined. We also had the opportunity to collect worms from each lamb for electron microscopic analysis of the effects of the varying concentration of CV on the morphology of the adult worms collected from the abomasum.
The project can be viewed in two parts, based on objectives:
Objective 1 is the development of a grain supplement incorporating cranberry vine. Cranberry vine (450 kg) was obtained from locally produced cranberry fields (A.D. Makepeace Company, Wareham, MA) during spring pruning of 2015. The vines were bagged in burlap sacks and air-dried at the Agronomy farm at the University of Rhode Island. The dried vines (200 kg) were chopped up using a feed mixer-grinder at Cornell University in Ithaca, NY. Based upon results from previous work in our research laboratory investigating the anthelmintic activity of a condensed tannin extract of cranberry vine, we fed a diet that contained either 100 or 200 grams of chopped cranberry vine to the lambs. These amounts correspond to 300 and 600 mg/ml in our in vitro studies with cranberry vine condensed tannin extract, concentrations that demonstrated anti-parasitic activity against larval and adult stages of Haemonchus contortus. We were not able to create a cranberry vine pellet because of difficulty acquiring access to a grinder that would process the CV fine enough for pelleting in the quantities needed. The cranberry vine was sufficiently palatable to the lambs without incorporating it into a pellet, so we decided to feed it without pelleting it. The nutritional composition of the cranberry vine diets were comparable to the control diets that were fed chopped alfalfa hay instead of cranberry vine.
Objective 2 is the determination of the anti-parasitic potential of CV. Twenty-one Dorset and Dorset cross-bred lambs, born and housed at the University of Rhode Island Peckham Farm, located in Kingston, R.I., were used for this study. An 18% crude protein commercially available pelleted supplement (Central CT Co-op, Manchester, CT) was offered to lambs shortly after birth through weaning. Thereafter, lambs were group fed 0.91 kg/lamb/day of grass hay and grain. Prior to the start of the study lambs were brought indoors and kept in indoor housing for the remainder of the study. Lambs continued to be group fed 0.91 kg/lamb/day of grass hay and grain and allowed free access to water and trace mineralized salt. Lambs were dewormed with two anthelmintics: levamisole hydrochloride (8.8 mg/kg BW, Levasole®, Schering-Plough Animal Health Corp., Union, NJ) and ivermectin (0.2 mg/kg BW, Ivomec®, Merial Inc., Duluth GA) and confirmed worm free prior to the start of the study. Lambs were orally administered 10,000 infective H. contortus L3 larvae. Fecal egg counts (FEC) were monitored one to two times per week and packed cell volume (PCV) and weight were monitored weekly throughout the trial. After the infection was allowed to mature (approximately 35 days), animals were stratified into dietary treatment groups based upon FEC and sex. There were two experimental diets containing 100 (CV100, n=7 lambs) or 200 (CV200, n=7 lambs) grams of dried cranberry vine per day and one control diet (n=7 lambs) containing chopped alfalfa hay. Lambs were on a treatment or control diet for a five-week experimental period. At the end of the five weeks, lambs were brought to the abattoir and the abomasum of each lamb was obtained for determination of worm burden. Worms were also collected for observation using scanning and transmission electron microscopy.
All infective larvae used in these assays were provided by Dr. A. Zajac, DVM, Ph.D, Parasitologist from VA-MD Regional College of Veterinary Medicine. Fecal egg counts were processed weekly as well as blood samples to determine weekly PCV and weekly weights were recorded. Rectal fecal samples were collected and FEC was determined by the modified McMaster technique (Whitlock, 1948) using 2 g feces and sodium nitrate flotation solution (Fecasol®, Vetoquinol U.S.A., Inc., Fort Worth, Texas). Each egg observed represented 50 eggs/gram of feces. Packed cell volume was determined by the micro-hematocrit centrifuge method. At the end of the five-week experimental feeding period, lambs were sacrificed and abomasa were collected for analyses. Recovery and enumeration of parasites was performed following the recommendations of the WAAVP Guidelines for evaluating efficacy of anthelmintics in ruminants (Wood et al 1995). The abomasum was opened along its greater curvature and five live female H. contortus worms, for electron microscopic evaluation, were collected from the abomasum of each of the 21 lambs at the abattoir and individually fixed in 2% glutaraldehyde solution in phosphate buffer (0.1 M, pH = 7.4). The abomasa were then washed with warm water and contents were collected. Content volume was brought up to two liters and a 10%-aliquot was taken and fixed with equal volume of 10% phosphate buffered formalin (PBF). Abomasal contents were examined and the remaining worms counted and collected.
Fecal egg count, weight, and PCV data was analyzed using GLM Procedure in SAS (SAS Institute Inc., Cary, NC) with repeated measures analysis of variance and the Tukey post-hoc test. The model included terms Treatment, Week, and Treatment*Week. Significance was defined as p ≤ 0.05. Worm burden data was analyzed using GLM Procedure in SAS and unpaired t-test procedure in Prism 5.0 for MAC OS X (GraphPad Software, San Diego, CA). Significance was defined as p ≤ 0.05.
Of the 21 animals processed for electron microscopic analysis, two lambs from the control group and five lambs from the CV200 group were investigated using two worms from each of those randomly selected animals. Worms were brought to the Morphology Service Laboratory at Virginia-Maryland College of Veterinary Medicine, Virginia Tech. Fixed worms were washed in 0.1 M Na Cacodylate buffer and post-fixed with 2% OsO4 in 0.1 M Na Cacodylate buffer. Worms were then dehydrated in a graded ethanol series (15%, 30%, 50%, 70%, 95%, 100%), and dried using Ladd Critical Point Dryer. The worms were then either gold plated to be observed using scanning electron microscopy, or set in a poly-bed resin to be cut (cross-sectioned) and viewed using transmission electron microscopy.
Whitlock, H. V. 1948. Some modifications of the mcmaster helminth egg-counting technique and apparatus. J. Counc. Sci. Res 21: 177-180. Wood, IB, Amaral NK, Bairden K et al. 1995.
Wood, I.B., N.K. Amaral, K. Bairden, J.L. Duncan, T. Kassai, J.B. Malone, Jr., J.A. Pankavich, R.K. Reinecke, O. Slocombe, S.M. Taylor and J. Vercruysse. 1995. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Vet Parasitol 58:181-213
The overall project did not progress as planned and did require us to make course corrections along the way, but the end goal of the project was still achieved and the lamb trial was completed. The project can be viewed in two parts, based on objectives:
Objective 1 is the development of a pelleted grain supplement incorporating cranberry vine. We were unable to develop a pelleted grain supplement because of a lack of equipment that could grind the cranberry vine into small enough particles to be pelletized in the quantities needed. With some small palatability tests, we were able to create a mixture of chopped alfalfa hay, molasses and CV that was enticing to the lambs. For this objective, we were able to develop a supplement containing varying concentrations of CV that lambs would readily consume.
Objective 2 is the determination of the anti-parasitic potential of CV. Using the CV diet, we were able to provide CV daily to lambs experimentally infected with H. contortus, for five weeks. An increase in FEC over the feeding period was observed in the Control and CV 100 lambs that did not occur in the CV200 lambs. Furthermore, there was an increase in PCV over time only in the CV200 lambs. Observations made using scanning and transmission electron microscopy indicate potential effects on the cuticle and feeding ability of the CV200 worms. Detailed results and discussion will be provided after the publication of this data.
The knowledge gained from this study has the potential to advance current knowledge in a way that will have a positive effect on whole-farm sustainability and productivity. Cranberry vine as an anthelmintic has the potential to provide an organic, natural alternative to synthetic commercial dewormers. If the CV is an effective treatment, this could potentially decrease producer reliance on commercial dewormers thus reducing the development of anthelmintic resistance of worms to the commercial dewormers and extending their efficacy. A reduced amount of drug residue on pastures would be environmentally beneficial for the soil microorganisms that aid in pasture productivity and nitrogen fixation. As a result, the use of CV may lead to an overall improvement in the whole-farm system, reducing environmental and health risks in agriculture. The GIN health concerns that are associated with putting lambs on pasture would also be addressed with the potential of this natural anthelmintic. Having lambs on pasture is a major health risk faced by small ruminant producers worldwide, establishing a crucial need for the knowledge gained through this study. Through reducing health risks like GIN infections, this would ultimately improve productivity due to lower worm burden, as well as reduce costs and increase the net farm income in conjunction with a decreased use of chemical dewormers.
Education & Outreach Activities and Participation Summary
The project results were shared with producers in the July 2015 and July 2016 field days focused on integrated gastrointestinal parasite control (URI Workshop & Field Day 2015 Flyer, URI Workshop & Field Day 2016 Flyer). At these field days, producers were updated on the results of ongoing parasite control research from our laboratory. The field days were well attended by 20-40 small ruminant producers each year. Producers were interested in and excited about the potential of cranberry vine as a parasite control method. The beneficiaries of this project include small ruminant producers, extension agents, researchers as well as undergraduate and graduate students. Providing this alternative method of GIN control will also assist farmers to realize increased earnings through reduced use of synthetic dewormers and increased animal productivity attributed to lower worm burden. An oral presentation of this data will be given at the American Association of Veterinary Parasitologists (AAVP) annual meeting in San Antonio, Texas, August 2016 (AAVP Abstract 2016). A manuscript of this data is being prepared for submission to the Journal of Veterinary Parasitology.
This project has the potential to beneficially change farm income by reducing the number of animals that are lost to GIN infections and increase production yield. It is difficult to estimate a numerical figure because herd health and susceptibility to these GIN infections vary between farms, sheep/goat breeds, and even individual animals. Ultimately the implementation of CV as a control agent of GIN infections has the potential to decrease farm costs, improve soil quality, and decrease labor; all of which can be achieved by decreasing the use of commercial dewormers. If CV were to be used as a GIN management tool, this could potentially be a viable option for farmers.
Farmer response to this project was positive, as they are eager to try this as an alternative method. With the ultimate goal of feeding pelleted CV, after presenting the results of this project, and showing the producers an example of what the cranberry vine would look like in pellet form, one producer responded to the presentation by yelling, “When can I get some pelleted cranberry vine for my farm?!”. The farmers are excited about the possibility of this potential parasite control method, because of its ease of application and adoption. As a feed supplement, this becomes a feasible option that requires little manual labor and is easy for the farmer to implement and adopt into their production system.
Areas needing additional study
While this project revealed much about the effects of cranberry vine on an experimental infection of Haemonchus contortus in lambs, there is more data that needs to be collected in vivo. Determination of the optimal feeding rate and timing of the consumption of CV is needed. Furthermore, an understanding of the mechanism of action of condensed tannins on gastrointestinal nematodes is essential and would greatly accelerate the discovery of effective control methods for GIN infections in small ruminants.