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Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats
Accepted Manuscript Title: Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats Author: A. Mechineni D.S. Kommuru S. Gujja J.A. Mosjidis J.E. Miller J.M. Burke A. Ramsay I. Mueller-Harvey G. Kannan J.H. Lee B. Kouakou T.H. Terrill PII: DOI: Reference:
S0304-4017(14)00338-0 http://dx.doi.org/doi:10.1016/j.vetpar.2014.06.002 VETPAR 7279
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
16-1-2014 30-5-2014 1-6-2014
Please cite this article as: Mechineni, A., Kommuru, D.S., Gujja, S., Mosjidis, J.A., Miller, J.E., Burke, J.M., Ramsay, A., Mueller-Harvey, I., Kannan, G., Lee, J.H., Kouakou, B., Terrill, T.H.,Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal
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nematode infections of growing goats
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A. Mechinenia, D.S. Kommurua, S. Gujjaa, J.A. Mosjidisb, J.E. Millerc, J.M. Burked, A.
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Ramsaye, I. Mueller-Harveye, G. Kannana, J.H. Leea, B. Kouakoua, and T.H. Terrilla*
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Fort Valley State University, Fort Valley, Georgia 31030, USA
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Auburn University, Auburn, Alabama 36849, USA
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USDA/ARS/DBSFRC, Booneville, Arkansas 72927, USA
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School of Agriculture and Policy, University of Reading, Reading RG6 6AT, UK
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Louisiana State University, Baton Rouge, Louisiana 70803, USA
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*Corresponding author. Tel.: 1-478-825-6814; fax: 1-478-825-6376.
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E-mail address: [email protected] (T.H. Terrill)
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ABSTRACT High prevalence of anthelmintic-resistant gastrointestinal nematodes (GIN) in
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goats has increased pressure to find effective, alternative non-synthetic control methods,
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one of which is adding forage of the high condensed tannin (CT) legume sericea
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lespedeza (SL; Lespedeza cuneata) to the animal’s diet. Previous work has demonstrated
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good efficacy of dried SL (hay, pellets) against small ruminant GIN, but information is
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lacking on consumption of fresh SL, particularly during the late summer-autumn period
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in the southern USA when perennial warm-season grass pastures are often low in quality.
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A study was designed to determine the effects of autumn (September-November)
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consumption of fresh SL forage, grass pasture (predominantly bermudagrass, BG;
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Cynodon dactylon), or a combination of SL+BG forage by young goats [intact male
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Spanish kids, 9 months old (20.7 ± 1.1 kg), n = 10/treatment group] on their GIN
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infection status.
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Three forage paddocks (0.40 ha) were set up at the Fort Valley State University
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Agricultural Research Station (Fort Valley, GA) for an 8-week trial. The goats in each
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paddock were supplemented with a commercial feed pellet at 0.45 kg/head/d for the first
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4 weeks of the trial, and 0.27 kg/head/d for the final 4 weeks. Forage samples taken at the
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start of the trial were analyzed for crude protein (CP), neutral detergent fiber (NDF), and
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acid detergent fiber (ADF) content, and a separate set of SL samples was analyzed for CT
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in leaves, stems, and whole plant using the benzyl mercaptan thiolysis method. Animal
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weights were taken at the start and end of the trial, and fecal and blood samples were
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collected weekly for determination of fecal egg counts (FEC) and packed cell volume
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(PCV), respectively. Adult GIN were recovered from the abomasum and small intestines
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of all goats at the end of the experiment for counting and speciation. The CP levels were
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highest for SL forage, intermediate for SL+BG, and lowest for BG forage samples, while
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NDF and ADF values were the opposite, with highest levels in BG and lowest in SL
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forage samples. Sericea lespedeza leaves had more CT than stems (16.0 vs 3.3 g/100 g
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dry weight), a slightly higher percentage of PDs (98 vs 94%, respectively) and polymers
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of larger mean degrees of polymerization (42 vs 18, respectively).
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There were no differences in average daily gain or blood PCV between the
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treatment groups, but SL goats had lower FEC (P < 0.05) than the BG or SL+BG forage
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goats throughout most of the trial. The SL+BG goats had lower FEC than the BG forage
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animals by the end of the trial (week 8, P < 0.05). The SL goats had lower numbers (P <
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0.05) of male H. contortus and tended to have fewer female (P <0.10) and total (P <
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0.07) H. contortus compared with the BG goats. The predominant GIN in all the goats
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was Trichostrongylus colubriformis (73% of total GIN).
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As a low-input forage with activity against pathogenic GIN (H. contortus), SL has
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a potential to reduce producers’ dependence upon synthetic anthelmintics and also to fill
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the autumn ‘window’ in good-quality fresh forages for goat grazing in the southern USA.
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1. Introduction
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Gastrointestinal nematode (GIN) infection is a major hindering factor in small
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ruminant production all over the world. Increased mortality and poor weight gains
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contribute to the majority of losses in the sheep and goat industry (Wanyangu and Bain,
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1994; Gatongi et al, 1997; Sharkhuu, 2001; Faye et al., 2003). Goats are even more 3
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susceptible than sheep to nematode infection (Tembely and Hansen, 1996). Goat
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production is an important industry in the southeastern USA because of availability of
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forages and also rising market demand, particularly for organically-produced meat
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(Robinson, 2004). However, GIN control, particularly of Haemonchus contortus, a
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pathogenic blood-feeder, is challenging in this region because the sub-tropical climate is
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ideal for growth of the free-living stages of GIN (infective L3 larvae) on pasture. This
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also applies to more northern parts of the USA that have periods of warm and humid
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weather, such as during the summer months. Infection with Trichostongylus
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colubriformis, and Teladorsagia circumcincta are also common in small ruminants
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during cooler periods of the year and can lead to production losses (Miller, 1996).
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Overuse of anthelminthic drugs to control small ruminant GIN has led to greatly
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increased incidence of anthelmintic resistance world-wide, making this an unreliable
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method to control parasites (Kaplan, 2004). Therefore, it is necessary to develop farm
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management systems that can maintain high levels of animal production with reduced
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reliance on anthelmintic intervention (Terrill et al., 2001; Shaik et al., 2006). There have
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been a number of reports on alternative (non-synthetic) methods to control parasites in
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small and large ruminants (Larsen, 1999; Kabagambe et al., 2000; Burke et al., 2004). A
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plant-based control strategy with great potential for on-farm application, particularly for
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small and limited resource producers, is feeding of forages containing condensed tannins
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(CT; Niezen et al., 2002; Shaik et al., 2006).
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Niezen et al. (1995) reported lower fecal egg counts (FEC) and worm burdens (T.
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colubriformis, T. circumcincta) in lambs grazing sulla (Hedysarum coronarium), a CT-
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containing legume, compared with alfalfa (Medicago sativa), which contains no CT. 4
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Other CT-containing plant species with reported efficacy against GIN (H. contortus, T.
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colubriformis, T. circumcincta) include big trefoil (Lotus pedunculatus; Niezen et al.,
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1998), birdsfoot trefoil (L. corniculatus; Heckendorn et al., 2007), and sainfoin
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(Onobrychus viciifolia; Heckendorn et al., 2006). In short-term studies, Min et al (2004;
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2005) reported reduced FEC in goats grazing sericea lespedeza (SL; Lespedeza cuneata)
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compared to a non-CT forage during the late spring-early summer period, while Shaik et
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al (2006) reported reduced FEC and lower numbers of adult GIN (H. contortus, T.
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colubriformis, T. circumcincta) in goats fed hay of SL compared with non-CT hay
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(bermudagrass - BG; Cynodon dactylon).
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Sericea lespedeza is a widely adapted, perennial warm-season legume that grows
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well on acidic and droughty soils in Georgia, USA (Hoveland et al., 1990). As a low-
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input forage, SL is an economical feed resource in the southeastern USA during the
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warmer (late spring/summer/early autumn) months, with similar animal performance as
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perennial warm-season grasses, like BG (Ball et al., 2002). Several studies have reported
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good efficacy of dried SL (hay, pellets) against GIN infection in goats during the late
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summer-autumn period in the southern United States (Shaik et al., 2004; Terrill et al.,
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2009; Gujja et al., 2013), but there have no reports on the potential anthelmintic effect of
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goats consuming fresh (grazed) SL during this period. The objective of this study was to
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determine the anthelminthic efficacy of fresh SL or perennial grass forage in the diet of
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naturally-infected goats during the late summer-early autumn period in Georgia.
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2. Materials and methods
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A trial with intact male Spanish kids was completed at the Fort Valley State
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University (FVSU) Agricultural Research Station, Fort Valley, GA during September,
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October, and November, 2011. All husbandry practices and experimental procedures
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used in the study were approved by the FVSU Animal Care and Use Committee.
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2.1. Forage paddocks
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Three paddocks (0.40 ha) were laid out in an area that had no previous grazing
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animals for over 12 months using a completely randomized design for three treatments:
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1) fresh SL forage (SL), 2) fresh grass (mixture of warm-season perennial species,
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predominantly BG) forage (BG), and 3) a combination of SL and BG forage (SL+BG).
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Spring and early summer growth on each of the paddocks was removed prior to the start
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of the trial, and grazing was initiated on late summer, autumn regrowth. The SL and
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SL+BG paddocks were fenced using electric netting (Electronet, Premier1 Supplies,
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Washington, IA) with a solar charger. The BG forage paddock had permanent fencing.
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Animals were set-stocked on each paddock throughout the study. To assure adequate
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nutrition, all animals were given supplemental pelleted feed (Mossey Creek feed,
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guaranteed analysis 14% crude protein, 4% crude fat, and 10% crude fiber; Mid-Georgia
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Farm Supply, Montezuma, GA) at 0.45 kg/head/d for the initial 4 weeks and 0.23
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kg/head/d for the remaining 4 weeks of the trial. The animals consumed all supplemental
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feed offered during the trial, and there was adequate forage leaf material available
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throughout the 8-week trial period in each paddock. All animals were allowed ad libitum
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access to water throughout the study period.
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2.2. Animals
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Thirty intact male Spanish kids (9 months old, 20.7 ± 1.1 kg) were used for this
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experiment. The goats were allowed to acquire a natural infection by grazing infected
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grass pasture for 4 weeks prior to the start of the trial. Animals were stratified by fecal
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egg count (FEC) and then randomly assigned to forage paddocks such that each of the
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goat groups (n = 10/paddock) had similar mean and standard deviation for total FEC.
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After initiation of the trial, blood and fecal samples were taken weekly from individual
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animals for FEC and packed cell volume (PCV) determination, respectively. Animal
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body weights (BW) were taken at the start and end of the trial. At the end of 8 weeks, the
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animals were transferred to the Fort Valley State University Meat Technology Center for
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slaughter, blood and fecal samples were taken, and GIN from the abomasum and small
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intestines were recovered for counting and speciation.
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2.3. Sampling and techniques
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2.3.1. Forages
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For forage quality determination, 10 random samples were taken from each
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paddock prior to starting the trial using a 30.48 cm2 quadrat, with all plant material within
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the quadrat cut to a 2.54 cm stubble. The samples from each paddock were composited,
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dried at 60º C for 48 hours, weighed, and then ground for quality analyses. Subsamples of 7
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the supplemental commercial pellets were collected for processing and analysis as well.
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A separate sample of SL was collected, immediately frozen, and then freeze-dried for CT
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analysis. A portion of the freeze-dried whole plant material was hand separated into
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leaves and stems, with leaf, stem, and whole plant samples ground for analysis.
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2.3.2. Forage sample analyses
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All oven-dried forage and feed pellet samples were ground to 1 mm particle size
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using a Cyclotec grinder (Foss, Eden Prairie, MN) and then analyzed for dry matter
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(DM), nitrogen (N), neutral detergent fiber (NDF), and acid detergent fiber (ADF).
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Analysis of N was completed using a Carbon/Nitrogen analyzer (Vario Max, Elementar
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Americas Inc., Mt. Laurel, NJ), with crude protein (CP) calculated as N x 6.25. Fiber
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analyses (NDF and ADF) were completed using the method of Van Soest et al (1991)
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using an ANKOM200/220 Fiber Analyzer (ANKOM Technology, Macedon, NY). Dry
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matter was determined using AOAC (1984) protocols. All forage quality data are
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presented on a DM basis.
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Leaf, stem, and whole plant samples of SL were freeze-dried and analyzed
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directly for CT content and composition using the benzyl mercaptan thiolysis method
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(Gea et al., 2011).
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2.3.3. Chemicals and standards
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Hydrochloric acid (36%), acetone (Analytical Reagent grade), dichloromethane
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(HPLC grade), and methanol (HPLC grade) were obtained from ThermoFisher Scientific 8
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(Loughborough, U.K.). (±)-Dihydroquercetin (98%) was obtained from Apin Chemicals
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(Abingdon, U.K.). Benzyl mercaptan (BM; 98%), (+)-catechin (C), (-)-epicatechin (EC),
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(-)-gallocatechin (GC), (-)-epigallocatechin (EGC) were obtained from Sigma-Aldrich
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(Poole, U.K.). Sephadex LH-20 was obtained from GE Healthcare (Little Chalfont, U.K.)
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and C18 mini-cartridges from Agilent Technologies (Wokingham, U.K.). Deionized and
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distilled water was obtained from a Milli-Q system (Millipore, Watford, U.K.).
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2.3.4. In situ thiolysis of condensed tannins
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Analysis of CT was performed according to a slightly modified method of Gea et
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al, (2011). Freeze-dried material of whole SL plants, leaves and stems (200 mg) was
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reacted with the thiolysis reagent (2 mL methanol, 1 mL of 3.3% HCl in methanol, and
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100 μL BM) as previously described. Methanol (1 mL) was added to the mixture. The
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sample was centrifuged at 4000 rpm for 3 min, and supernatant (1 mL) was transferred
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into another screw cap glass tube. Distilled water (9 mL) was added to this supernatant.
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After thorough mixing, the solution was added to a prepacked C18-mini-cartridge and
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conditioned as in Gea et al. (2011). Methanol (2.5 mL) was added to elute thiolysis
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reaction products under gravity and the internal standard, dihydroquercetin in methanol
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(500 μL; 0.1 mg/ml), was added into the collection tubes. The reaction products were
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analyzed by liquid chromatography-mass spectrometry (LC-MS; see below).
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In addition, free flavanol monomers were analyzed by LC-MS directly in plant
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samples using the above ‘thiolysis reagent’, where the HCl-methanol (1 mL) and BM
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(100 μL) reagents were replaced with methanol (1100 μL). 9
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2.3.8. Analysis of thiolysis reaction products by liquid chromatography-mass
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spectrometry (LC-MS)
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Flavan-3-ols and their BM adducts were identified by LC-MS analysis on a HPLC
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Agilent 1100 series system and API-ES instrument Hewlett Packard 1100 MSD Series
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(Agilent Technologies, Waldbronn, Germany). Samples (20 μL) were injected into a
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HPLC system connected to an ACE C18 column (3 μm; 250 x 4.6 mm; Hichrom Ltd;
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Theale; U.K.) fitted with a guard column at room temperature. The HPLC system
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consisted of a G1379A degasser, G1312A binary pump, a G1313A ALS autoinjector, a
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G1314A VWD UV detector, a G1316A column oven, and a personal computer with
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LC/MSD Agilent ChemStation version A 10.01 software. The flow rate was 0.75 mL/min
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using 1% acetic acid in water (solvent A) and HPLC-grade methanol (solvent B). The
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following gradient program was employed: 0-52 min, 36% B; 52-60 min, 36-50% B
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linear; 60-65 min, 50-100% B linear; 65-73 min, 100-0% B; 73-80 min, 0% B.
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Chromatograms were recorded at 280 nm, and MS spectra were recorded in the negative
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ionization scan mode between m/z 100 and 1000 using the following conditions: capillary
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voltage, 3000 V; nebulizer gas pressure, 35 psig; drying gas, 12 mL/min; and dry heater
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temperature, 350 °C. The concentrations of free flavanols were subtracted from
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concentrations of terminal flavanols, which were released during thiolysis.
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This provided information on the tannin composition in terms of % terminal and
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% extension flavanol units; it also allowed calculation of mean degree of polymerization
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(mDP), % procyanidins (PC) and prodelphinidins (PD), and % cis- and trans flavanols 10
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according to Gea et al. (2011).
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2.4. Animal samples
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2.4.1. Blood samples
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Blood samples were collected via jugular venipuncture into 3 mL vacutainer
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blood tubes containing K2EDTA. Samples were returned to the lab for immediate
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determination of packed cell volume (PCV) in duplicate using a microhematocrit
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centrifuge and reader. Micro-hematocrit capillary tubes were filled with whole blood,
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sealed, and then centrifuged for 10 minutes at 9245 x g in a hematocrit centrifuge (IEC
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Model Mb, Fisher Scientific). The hematocrit values were read directly from the
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centrifuge scale.
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2.4.2. Fecal samples
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Fecal samples were collected directly from rectum of all animals weekly during
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the trial to determine FEC using the modified McMaster technique (Whitlock, 1948). The
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results were expressed as eggs per gram of feces (EPG) by the following formula:
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Average number of eggs in 2 counting chambers x 100.
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2.4.2. Recovery and counting of adult nematodes At the end of the trial, the goats were slaughtered at the USDA-approved abattoir at the Fort Valley State University Agricultural Research Station.
Fecal and blood
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samples were taken from individual animals for FEC and PCV determination,
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respectively, and adult worms were recovered from the abomasum and small intestines as
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described by Shaik et al (2006). After slaughter, the abomasum and small intestine of
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each goat was ligated, opened, and the contents washed into plastic buckets. The contents
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were brought up to 3 L with tap water, thoroughly mixed, and then two 5% aliquots (150
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mL) taken into 250 mL storage containers. Approximately 100 mL of formalin (10%)
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were added to each aliquot as a preservative for both abomasal and small intestinal
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samples. These were brought up to 3 L and subsampled and preserved as described
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previously. For processing, the adult worms in both (abomasal and small intestinal)
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aliquots were washed on a mesh screen (53 microns) with tap water, the formalin
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discarded, and the nematodes recovered into a 50 mL centrifuge tube. All the nematodes
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in the tube were then counted and identified to species and sex using a Leica Zoom 2000
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phase contrast microscope (Leica Microsystems Inc., Chicago, IL).
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2.5. Statistical analyses
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Fecal egg count and blood PCV data were analyzed using repeated measures
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analysis as a completely randomized design (SAS, 2008). The model included treatment,
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day as a repeated measure, and the interaction. When treatment effects were different at
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P < 0.05, means were separated using LSD test. Adult GIN, beginning and ending BW,
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and forage quality data were analyzed as a completely randomized design using the GLM
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procedure of SAS. The FEC and adult GIN data were log-transformed (log FEC + 1)
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prior to statistical analysis. Fecal egg count and adult GIN data were reported as least
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squares means, with statistical inferences based upon log-transformed data analysis. 12
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Condensed tannin analyses were only completed for SL samples, so these data were not
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subjected to statistical analysis.
3. Results
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3.1. Chemical composition of pasture and pellet samples
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The CP, NDF, and ADF values for all forage samples are shown in Table 1. The
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CP values were highest for the SL, intermediate for SL+BG, and lowest for the BG
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forage, while the fiber values were the opposite, with lowest and highest values for the
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SL and BG forages, respectively. The commercial pellet had 19.6, 15.0, and 9.4% CP,
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NDF, and ADF, respectively.
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Leaves of SL had much higher tannin content than stems (16.0 vs 3.3 g/100 g dry
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weight) (Table 2). Leaf CT were also much larger than stem CT, with mDP being 42 for
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leaves and 18 for stems. Both CT are almost pure PD: i.e. 98% of CT are PDs in leaves
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compared to 94% in stems. Both leaves and stems had small amounts of PC. The cis-
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flavanol proportion is slightly higher in leaves (91%) than stems (84%). Whole plant
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samples were intermediate in total CT content, mDP, PC, PD, and proportion of cis-
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flavanols (Table 2).
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SL samples contained a small amount of free or non-tannin epigallocatechin
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(0.2%) and 0.01% of gallocatechin (Table 3). The flavanols in terminal and extension
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units were mostly epigallocatechin (87.4%), followed by gallocatechin (9.6%) and
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epicatechin (2.6%). Catechin was only detected in extension units (0.5%). 13
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3.2. Fecal egg count Fecal egg counts were similar between the three treatment groups at the start of
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the trial (week 0). Two weeks after initiation of the trial, FEC from goats consuming SL
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forage were lower (P < 0.04) than for animals grazing the other two pastures (BG and
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SL+BG) (Figure 1). At the 3rd and 4th week samplings, FEC results were not different
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among treatment groups, but starting from the 5th week through the end of the trial, FEC
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of the SL goats were significantly lower (P < 0.02) compared with the other two
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treatment groups. Compared to control goats, the SL animals had 82.4% lower FEC by
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week five, and this increased to 95.4% reduction by the end of the trial. The FEC values
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for the SL+BG treatment goats were not different from controls until the final week of
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the trial, when the SL+BG group FEC were 71.5% lower (P < 0.03).
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3.3. Packed cell volume (PCV)
The PCV values for all the goats were initially low (ranged from 14-17%) and
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increased throughout the trial (final sampling values ranged from 27-28%). There was no
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significant difference (P = 0.59) among the three treatment groups throughout the
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experiment (Figure 2).
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3.4. Body weights
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Initial and final body weights and ADG for all the goats are presented in Table 4.
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There was no effect of pasture forage type on final live weight or gain per day in the
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goats. 14
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3.5. Adult nematodes The adult nematode worm species recovered from abomasal samples were H.
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contortus and T. circumcincta. Total GIN and number of males and females counted for
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each nematode species are presented in Table 5. Number of adult H. contortus males was
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lower (P < 0.05) in goats consuming SL pasture forage compared with BG pasture
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animals, and female and total H. contortus tended to be lower (P < 0.09, P < 0.07,
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respectively) in the SL groups compared with BG goats. Although there was no
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significant difference in small intestinal nematodes (T. colubriformis) between the
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groups, total and male T. colubriformis numbers tended to be lower (P < 0.11, P < 0.06,
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respectively) in the SL compared with the mixed forage (BG+SL) goats. Overall, there
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was a trend (P < 0.11) for lower total worms in the SL group compared with the SL+BG
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goats.
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4. Discussion
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4.1. Condensed tannins in sericea lespedeza Sericea lespedeza has a surprisingly high CT content (12.5 g/100 g) and a
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relatively rare CT composition (Table 2). SL tannins were almost pure PDs (97%) of high
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molecular weight (mDP = 33, which corresponds to 10,100 Daltons) and also had a high
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proportion of cis-flavanols (90%), which is due to a preponderance of epigallocatechin
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(87%) and gallocatechin (10%) units (Table 3). The same tannin method has previously
333
been used to screen sainfoin accessions (Stringano et al., 2012). This germplasm 15
Page 15 of 29
collection covered CT contents ranging from 0.6 to just 2.8 g/100 g dry weight. Only 16
335
out of 37 accessions had tannins with more than 80% PDs, just 1 accession had more than
336
90% PDs and 15 accessions had polymers with mDP-values above 30. The finding that
337
leaves not only had a higher CT concentration than stems, but also a higher PD
338
percentage and larger CT polymers (mDP-value) agrees with results from sainfoin
339
(Theodoridou et al., 2010 and 2011).
cr
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334
341
us
340
4.2. Anti-parasitic effects
The most effective treatment in this study against GIN infection in goats,
343
particularly for H. contortus, was fresh SL forage, which confirms previous reports on the
344
anthelmintic properties of this plant in fresh and dried forms for small ruminants (Min et
345
al., 2004; 2005; Shaik et al., 2006; Terrill et al., 2007; 2009). Consumption of mixed
346
SL+BG fresh forages also had some effect in reducing GIN infection in goats compared
347
with grass (BG) only, but the effect took longer (Figure 1), similar to results of Burke et
348
al (2012a) with lambs grazing mixed grass and SL.
350 351
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an
342
4.2.1. Fecal egg counts
The initial reduction (P < 0.05) in FEC in goats consuming fresh SL compared
352
with grass (BG) (33% reduction) or SL+BG forage (56%) occurred two weeks after the
353
animals were turned onto the pastures, and these differences increased throughout the
354
trial, to 95 and 84%, respectively, by the final sample date. The goats consuming SL+BG
355
forage had 41% lower FEC than control (BG) animals by week 5 of the study, and these
356
differences also increased throughout the remainder of the trial, but reached significance 16
Page 16 of 29
(P < 0.05) only at the final sample date (71% reduction). The FEC reductions attained by
358
consuming fresh SL alone or in mixture with BG in autumn confirm past reports for goats
359
grazing SL in summer (Min and Hart, 2003; Min et al., 2005) or fed SL in dried forms
360
(Shaik et al., 2006; Terrill et al., 2009). In contrast, Burke et al. (2012a) reported no
361
effect of summer-grazed SL on predominantly Trichostrongylus spp. infected goats, but
362
reduced (P < 0.05) FEC in lambs (predominantly H. contortus in control lambs) grazing
363
SL alone or in a mixture with BG compared with grass pastures only (Burke et al.,
364
2012b). The delay in FEC reduction for the goats consuming fresh SL+BG forage
365
compared to BG alone in the current investigation may be due to several factors. Based
366
upon daily observation, the goats in the mixed SL+BG pasture initially grazed the non-
367
SL components for the first 2-3 weeks, after which they grazed the BG and SL more
368
evenly. In the SL paddock (which also had a small amount of grasses along the fence
369
lines), the goats started grazing the SL plants within 2-3 days. In previous reports,
370
reduction in FEC due to SL feeding was slower during times of the year when H.
371
contortus was not the primary GIN, such as in the autumn or winter months (Shaik et al.,
372
2004; Terrill et al., 2009). The current study was completed during mid-September to
373
mid-November with goats that had a non-H. contortus-dominant infection (73% T.
374
colubriformis). This may also explain the lack of a treatment effect on PCV values in the
375
animals, as anemia scores have been reported to be less affected by SL when H. contortus
376
is not the predominant GIN (Terrill et al., 2009). However, overall low numbers of adult
377
H. contortus in all the animals is more likely the reason for the lack of difference in
378
anemia scores between treatment groups (Figure 2). In the current study, differences in
Ac ce p
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357
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Page 17 of 29
379
GIN infection or FEC reduction could be due to the higher quality of SL relative to BG,
380
although forage quality was only measured initially. Regardless of the type of GIN infection, a potential long-term benefit of reduced
382
fecal egg shedding in goats consuming fresh SL forage in autumn may be reduced
383
subsequent pasture infectivity (number of GIN L3 per unit pasture DM), such as in the
384
following spring.
cr
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381
386
us
385
4.2.2. Adult nematodes
The goats consuming fresh SL forage had 48% lower numbers of adult H.
388
contortus in the abomasum (P < 0.07) compared with control animals, with greater
389
differences in males (P < 0.05) than in females (P < 0.10). These results contrast with
390
those of Shaik et al. (2006), who reported a greater reduction effect of SL hay on female
391
than male GIN, including H. contortus. Reasons for the differential sex effect in the
392
current study are unclear, but the most important effect from the standpoint of reducing
393
egg production is obviously with the females. The SL goats also had 34% fewer numbers
394
of T. circumcincta than controls, and 44% fewer total abomasal (H. contortus + T.
395
circumcincta) than small intestinal worms, but the differences were not significant (Table
396
5). There have been few reports of effects of grazed SL on worm burdens in small
397
ruminants, but reports from sheep and goat feeding trials with dried SL (ground and
398
unground hay, pellets) have found greater effects on H. contortus adults than for other
399
GIN species (Shaik et al., 2006; Lange et al., 2006; Terrill et al., 2007; 2009). Shaik et al
400
(2006) reported reductions of 70, 26, and 40% for adult H. contortus, T. circumcincta and
401
T. colubriformis, respectively, in animals fed SL long hay as opposed to BG hay as 75%
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Page 18 of 29
of their ration. In the current study, initially goats would be expected to receive
403
approximately 30% of their diet from forage if consuming 3% of BW. Later, this
404
increased to about 60% on those same principles (CP and energy would have been
405
greatest for goats consuming SL). Regardless, the overall reduction effect of SL on adult
406
worms was against abomasal as opposed to small intestinal GIN.
ip t
402
Sericea lespedeza has been reported to be an effective bioactive forage against
408
small ruminant GIN when used in fresh (grazed) or dried forms (Min et al., 2005; Shaik
409
et al., 2006). These effects have been attributed to both direct effects on the adult worm
410
(killing the worm or reducing female fecundity; Min et al., 2005; Shaik et al., 2006) or
411
indirect effects of improving the nutritional status of the host by protecting protein from
412
rumen degradation and increasing amino acid flow to the small intestines (Waghorn et
413
al., 1997), allowing increased resilience to GIN infection (Moore et al., 2008). Definitive
414
differences between direct and indirect effects of SL on GIN (CT versus increased
415
protein effect) could not be separated because of the experimental design in the current
416
investigation. There is evidence for direct effects of fresh SL on GIN in the current
417
investigation (reduced FEC and H. contortus numbers), but there were no treatment
418
differences in animal performance (weight gains). All animals gained from 80 to 110 g/d
419
throughout the trial, suggesting that the goats all had adequate nutrition, which allowed
420
them to continue to be moderately productive despite differences in GIN status.
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421 422
5. Conclusions
423
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Page 19 of 29
Autumn consumption of fresh SL forage reduced FEC and H. contortus numbers
425
in goats compared with perennial warm-season grass (BG) forage, while giving
426
comparable animal performance. It is possible that SL is particularly effective at
427
controlling H. contortus due to its unusual tannins, which are almost pure prodelphinidins
428
of relatively high molecular weight. A long-term benefit of autumn grazing of SL pasture
429
may be reduced subsequent pasture infectivity due to lower numbers of GIN eggs on
430
pasture. As a low-input crop, and with its potential use as a natural anti-parasitic agent,
431
SL is an economical forage for small ruminant production in the southern USA, while
432
potentially reducing farmers’ dependence upon synthetic anthelmintics.
an
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Acknowledgements
This research was supported by the USDA NIFA Organic Research and
d
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Education Initiative (Project no. 2010-51300-21641). A. Ramsay acknowledges financial
437
support from the European Union (PITN-GA-2011-289377, ‘LegumePlus’).
439 440 441 442 443 444 445 446 447 448 449 450 451 452
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References
AOAC, 1984. Official Methods of Analysis (14th Ed.). Association of Official Analytical Chemists, Washington, D.C. Ball, D.M., Hoveland, C.S., Lacefield, G.D., 2002. Southern Forages, Third edition, Graphic Communications Corporation, Lawrenceville, GA, 322p. Burke, J.M., Miller, J.E., Mosjidis, J.A., Terrill, T.H., 2012a. Use of a mixed sericea lespedeza and grass pasture system for control of gastrointestinal nematodes in lambs and kids. Vet. Parasitol. 186, 328-336. Burke, J.M., Miller, J.E., Mosjidis, J.A., Terrill, T.H., 2012b. Grazing sericea lespedeza for control of gastrointestinal nematodes in lambs. Vet. Parasitol. 186, 507-512. Burke, J.M., Miller, J.E., Olcott, D.D, Olcott, B.M, Terrill, T.H., 2004. Effect of copper oxide wire particles dosage and feed supplement level on Haemonchus contortus infection in lambs. Vet. Parasitol. 123, 235-243. 20
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Faye, D., Leak, S., Nouala, S., Fall, A., Losson, B., Geerts, S., 2003. Effects of gastrointestinal helminth infections and plane of nutrition on the health and productivity of F1 (West African Dwarf × Sahelian) goat crosses in The Gambia. Small Rumin. Res. 50, 153-161. Gatongi, P.M., Scott, M.E., Ranjan, S., Gathuma, J.M., Munyua, W.K., Cheruiyot, H., Prichard, R.K., 1997. Effects of three nematode anthelmintic treatment regimes on flock performance of sheep and goats under extensive management in semiarid Kenya. Vet. Parasitol. 68, 323-336. Gea, A., Stringano, E., Brown, R.H., Mueller-Harvey, I., 2011. In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high throughput germplasm screening. J. Agric. Food Chem. 59, 495-503. Gujja, S., Terrill, T.H., J.A. Mosjidis, J.E. Miller, A. Mechineni, D.S. Kommuru, S.A. Shaik, B.D. Lambert, N.M. Cherry, and J.M. Burke. 2013. Effect of supplemental sericea lespedeza leaf meal pellets on gastrointestinal nematode infection in grazing goats. Vet. Parasitol. 191, 51-58.
468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497
Heckendorn F., Haring, D.A., Maurer, V., Zinsstag, J., Lanfhans, W., Hertzberg, H. 2006. Effect of sainfoin (Onobrychis viciifolia) silage and hay on established populations of Haemonchus contortus and Cooperia curticei in lambs. Vet. Parasitol. 142, 293-300. Heckendorn F., Haring, D.A., Maurer, V., Senn, M., Hertzberg, H. 2007. Individual administration of three tanniferous forage plants artificially infected with Haemonchus contortus and Cooperia curticei. Vet. Parasitol. 146, 123-134. Hoveland, C.S., Windham, W.R., Boggs, D.L., Durham, R.G., Calvert, G.V., Newsome, J.F., Dobson, Jr., J.W., Owsley, M., 1990. Sericea production in Georgia. Research Bulletin 393; The Georgia Agricultural experiment stations, College of Agriculture, The University of Georgia. Kabagambe, E.K., Barras, S.R., Li., Y., Pena, M.T., Smith, W.D., Miller, J.E., 2000. Attempts to control haemonchosis in grazing ewes by vaccination with gut membrane proteins of the parasite. Vet. Parasitol. 92, 15-23. Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol. 20, 477-481. Lange, K., Olcott, D.D., Miller, J.E., Mosjidis, J.A., Terrill, T.H., Burke, J.M., Kearney, M.T., 2006. Effect of sericea lespedeza (Lespedeza cuneata) fed as hay, on natural and experimental Haemonchus contortus infections in lambs. Vet. Parasitol. 141, 273-278. Larsen, M., 1999. Biological control of helminths. Int. J. Parasitol. 29, 139-146. Miller, J.E. 1996. Controlling goat parasites in the southeast. pp. 80-82 In: Proc. Southeast Reg. Meat Goat Prod. Sym., 21-24 February, 1996, Florida A&M University, Tallahassee, FL. Min, B.R., Hart, S.P., 2003. Tannins for suppression of internal parasites. J. Anim. Sci. 81 (E-Suppl. 2), 102-109. Min, B.R., Pomroy, W.E., Hart, S.P., Sahlu, T., 2004. The effect of short-term consumption of a forage containing condensed tannins on gastro-intestinal nematode parasite infections in grazing wether goats. Small Rumin. Res. 51, 279283.
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Min, B.R., Hart, S.P., Miller, D., Tomita, G.M., Loetz, E, Sahlu, T., 2005. The effect of grazing forage containing condensed tannins on gastro-intestinal parasite infection and milk composition in Angora does. Vet. Parasitol. 130, 105-113. Moore, D.A., Terrill, T.H., Kouakou, B., Shaik, S.A., Mosjidis, J.A., Miller, J.E., Vanguru, M., Kannan, G., Burke, J.M., 2008. The effects of feeding sericea lespedeza hay on growth rate of goats naturally infected with gastrointestinal nematodes. J. Anim. Sci. 86, 2328-2337. Niezen, J.H., Waghorn, T.S., Charleston, W.A.G., Waghorn, G.C., 1995. Growth and gastrointestinal nematode parasitism in lambs grazing either lucerne (Medicago sativa) or sulla (Hedysarum coronarium) which contains condensed tannins. J. Agric. Sci. 125, 281-289. Niezen, J. H., Robertson, H.A., Waghorn, G.C., Charleston, W.A.G., 1998. Production, fecal egg counts and worm burdens of ewe lambs which grazed six contrasting forages. Vet. Parasitol. 80, 15–27. Niezen, J.H., Charleston, W.A.G., Robertson, H.A., Shelton, D., Waghorn, G.C., Green, R., 2002. The effect of feeding sulla (Hedysarum coronarium) or Lucerne (Medicago sativa) on lamb parasite burdens and development of immunity to gastrointestinal nematodes. Vet. Parasitol. 105, 229-245. Robinson, J., 2004. Pasture Perfect: The Far-reaching Benefits of Choosing Meat, Eggs, and Dairy Products from Grass-fed Animals. Vashon Island Press, Vashon, WA, 160 pp. SAS Institute, 2008. SAS/STAT Software: changes and enhancements. Release 9.2, SAS Technical Report, SAS Institute, Cary, NC. Shaik, S.A., Terrill, T.H., Miller, J.E., Kouakou, B., Kannan, G., Kallu, R.K., Mosjidis, J.A., 2004. Effects of feeding sericea lespedeza hay to goats infected with Haemonchus contortus. SAJ Anim. Sci. 34, 234-236. Shaik, S.A., Terrill, T.H., Miller, J.E., Kouakou, B., Kannan, G., Kaplan, R.M., Burke, J.M., Mosjidis, J.A., 2006. Sericea lespedeza hay as a natural deworming agent against gastrointestinal nematode infection in goats. Vet. Parasitol. 139, 150-157. Sharkhuu, T., 2001. Helminths of goats in Mongolia. Vet. Parasitol. 101, 161-169. Stringano, E., Hayot Carbonero, C., Smith, L.M.J., Brown, R.H., Mueller-Harvey, I., 2012. Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection. Phytochem. 77, 197-208. Tembely, S., Hansen, J.W., 1996. Helminth Diseases of Small Ruminants in the Tropics: A Review of Epidemiology and Control Strategies. 22-25 April 1996; Bogor, Indonesia, 123-127. Terrill, T.H., Kaplan, R.M., Larsen, M., Samples, O.M., Miller, J.E., Gelaye, S., 2001. Anthelmintic resistance on goat farms in Georgia: efficacy of anthelmintics against gastrointestinal nematodes in two selected goat herds. Vet. Parasitol. 97, 261-268. Terrill, T.H., Mosjidis, J.A., Moore, D.A., Shaik, S.A., Miller, J.E., Burke J.M., Muir, J.P., Wolfe, R., 2007. Effect of pelleting on efficacy of sericea lespedeza hay as a natural dewormer in goats. Vet. Parasitol. 146, 117-122.
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498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540
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566 567 568
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d te
565
Terrill, T.H., Dykes, G.S., Shaik, S.A., Miller, J.E., Kouakou, B., Kannan, G., Burke, J.M., Mosjidis, J.A., 2009. Efficacy of sericea lespedeza hay as a natural dewormer in goats: Dose titration study. Vet. Parasitol. 163, 52-56. Theodoridou, K., Aufrère, J., Andueza, D., Pourrat, J., Le Morvan, A., Stringano, E., Mueller-Harvey, I., Baumont, R. 2010. Effects of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep. Anim. Feed Sci. Technol. 160, 23-38. Theodoridou, K., Aufrère, J., Andueza, D., Le Morvan, A., Picard, F., Stringano, E., Pourrat, J., Mueller-Harvey, I., Baumont, R., 2011. Effect of plant development during first and second growth cycle on chemical composition, condensed tannins and nutritive value of three sainfoin (Onobrychis viciifolia) varieties and lucerne. Grass For. Sci. 66, 402–414. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597. Waghorn, G.C., Ulyatt, M.J., John, A., Fisher, M.T., 1987. The effect of condensed tannins on the site of digestion of amino acids and other nutrients in sheep fed on lotus. Brit. J. Nutr. 57, 115–126. Wanyangu, S.W., Bain, R.K., 1994. The impact of helminths in small ruminant production in tropical Africa. Kenyan Vet. 18, 104-106. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg counting technique apparatus. J. Coun. Sci. Ind. Res. 21, 177-180.
Ac ce p
541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564
23
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568
Table 1. Chemical composition of fresh sericea lespedeza (SL), bermudagrass (BG), and
569
a mixture of SL+BG forage (All values expressed on dry matter basis). Constituent1
570
NDF %
BG
6.3
71.7
SL
14.7
35.4
SL+BG
10.8
56.0
1
ADF %
ip t
CP %
cr
35.2
us
Forage type
25.2 33.4
CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber.
an
571 572
Table 2. Analysis of tannin content (g/100 dry weight) and composition of sericea
574
lespedeza (SL) in terms of mean degree of polymerization (mDP), percentage of
575
prodelphinidins (PD) and procyanidins (PC), percentage of cis- and trans-flavanols (±
576
standard error).
d
te
Ac ce p
577
M
573
Plant material CT content
mDP
PD
PC
Cis
Trans
%
%
%
%
(SL)
(g/100g)
Whole plant
12.5±0.8
32.8±0.9 96.9±0.1 3.1±0.1 90.0±0.0 10.0±0.0
Leaves
16.0±1.8
41.9±1.8 98.0±0.1 2.0±0.1 90.8±0.0 9.17±0.0
Stems
3.33±0.3
17.8±0.8 93.6±0.1 6.4±0.1 83.8±0.2 16.2±0.2
578
24
Page 24 of 29
ip t cr
us
Table 3. Flavanol composition of free monomers and tannins in the whole plant, in leaves and stems of sericea lespedeza (SL) (±
Plant material
Flavanol monomers
(SL)
% GC
% EGC
M an
standard error).
Terminal units % GC
% EGC
%C
% EC
Extension units (BM1-adducts) % GC-BM % EGC-BM
% C-BM
% EC-BM
0.01±0.004 0.19±0.02 0.40±0.03 2.20±0.10
ND
0.46±0.11
9.18±0.03
85.2±0.10
0.46±0.02
2.15±0.01
Leaves
0.02±0.005 0.22±0.02 0.36±0.02 1.88±0.14
ND
0.16±0.01
8.51±0.02
87.2±0.29
0.29±0.01
1.57±0.15
ND
0.49±0.02
14.9±0.22
73.6±0.03
0.75±0.05
5.20±0.03
ND
ND = none detected 1
0.13±0.02 0.61±0.02 4.54±0.24
ce pt
Stems
ed
Whole plant
Ac
BM = benzyl mercaptan; GC = gallocatechin; EGC = epigallocatechin, C = catechin; EC = epicatechin.
25
Page 25 of 29
ip t
Table 4. Body weights and average daily gain (ADG) in goats consuming fresh sericea lespedeza (SL), bermudagrass (BG), or a mixture of SL+BG forage (± standard error).
Forage type
cr
Body weight (kg)
Final live weight
ADG (g)
BG
21±1.2
26±1.8
85±14.1
SL
20±1.1
26±1.7
108±13.3
BG + SL
21±1.0
25±1.6
81±12.6
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Initial live weight
26
Page 26 of 29
Table 5. Total male and female nematodes (± standard error) recovered from abomasum and small intestines of goats consuming fresh sericea lespedeza (SL), bermudagrass
Adult nematodes
BG
SL
Abomasum (total)
1468±411
817±390
H. contortus
1206±381
627±362
898±362
Male
509±193b1
us
Pasture treatment
ip t
(BG), or a mixture of SL+BG forage.
309±183a
391±183ab
318±191
507±191
113±42
119±42
53±38
51±38
60±15
68±15
3594±505
4725±505
3974±532
3594±505
4725±505
1696±228
1604±217
2184±217
Female
2279±310
1988±295
2621±295
Total worms
5442±720
4411±683
5780±683
T. circumcincta
170±44
Male
116±40
Female
54±15
T. colubriformis
1
1055±390
cr
d
Ac ce p
Male
3974±532
te
Small intestine (total)
an
697±202
M
Female
BG + SL
Row means with differing superscripts differ at P < 0.05. If no superscripts, then not
different (P > 0.05).
27
Page 27 of 29
ip t cr us an
M
Figure 1. The effect of consuming fresh sericea lespedeza (SL), bermudagrass (BG) or
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parasitized goats.
d
SL+BG mix forage on gastrointestinal nematode eggs per gram (EPG) of feces from
28
Page 28 of 29
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Figure 2. The effect of consuming fresh sericea lespedeza (SL), bermudagrass (BG) or
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d
SL+BG mix forage on blood packed cell volume (PCV) of parasitized goats.
29
Page 29 of 29
S0304-4017(14)00338-0 http://dx.doi.org/doi:10.1016/j.vetpar.2014.06.002 VETPAR 7279
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
16-1-2014 30-5-2014 1-6-2014
Please cite this article as: Mechineni, A., Kommuru, D.S., Gujja, S., Mosjidis, J.A., Miller, J.E., Burke, J.M., Ramsay, A., Mueller-Harvey, I., Kannan, G., Lee, J.H., Kouakou, B., Terrill, T.H.,Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal nematode infections of growing goats, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of fall-grazed sericea lespedeza (Lespedeza cuneata) on gastrointestinal
2
nematode infections of growing goats
3
A. Mechinenia, D.S. Kommurua, S. Gujjaa, J.A. Mosjidisb, J.E. Millerc, J.M. Burked, A.
4
Ramsaye, I. Mueller-Harveye, G. Kannana, J.H. Leea, B. Kouakoua, and T.H. Terrilla*
ip t
1
cr
5 a
Fort Valley State University, Fort Valley, Georgia 31030, USA
7
b
Auburn University, Auburn, Alabama 36849, USA
8
c
9
d
10
e
13 14
an
M
USDA/ARS/DBSFRC, Booneville, Arkansas 72927, USA
d
School of Agriculture and Policy, University of Reading, Reading RG6 6AT, UK
te
12
Louisiana State University, Baton Rouge, Louisiana 70803, USA
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6
15
*Corresponding author. Tel.: 1-478-825-6814; fax: 1-478-825-6376.
16
E-mail address: [email protected] (T.H. Terrill)
17
1
Page 1 of 29
17
ABSTRACT High prevalence of anthelmintic-resistant gastrointestinal nematodes (GIN) in
19
goats has increased pressure to find effective, alternative non-synthetic control methods,
20
one of which is adding forage of the high condensed tannin (CT) legume sericea
21
lespedeza (SL; Lespedeza cuneata) to the animal’s diet. Previous work has demonstrated
22
good efficacy of dried SL (hay, pellets) against small ruminant GIN, but information is
23
lacking on consumption of fresh SL, particularly during the late summer-autumn period
24
in the southern USA when perennial warm-season grass pastures are often low in quality.
25
A study was designed to determine the effects of autumn (September-November)
26
consumption of fresh SL forage, grass pasture (predominantly bermudagrass, BG;
27
Cynodon dactylon), or a combination of SL+BG forage by young goats [intact male
28
Spanish kids, 9 months old (20.7 ± 1.1 kg), n = 10/treatment group] on their GIN
29
infection status.
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Three forage paddocks (0.40 ha) were set up at the Fort Valley State University
31
Agricultural Research Station (Fort Valley, GA) for an 8-week trial. The goats in each
32
paddock were supplemented with a commercial feed pellet at 0.45 kg/head/d for the first
33
4 weeks of the trial, and 0.27 kg/head/d for the final 4 weeks. Forage samples taken at the
34
start of the trial were analyzed for crude protein (CP), neutral detergent fiber (NDF), and
35
acid detergent fiber (ADF) content, and a separate set of SL samples was analyzed for CT
36
in leaves, stems, and whole plant using the benzyl mercaptan thiolysis method. Animal
37
weights were taken at the start and end of the trial, and fecal and blood samples were
38
collected weekly for determination of fecal egg counts (FEC) and packed cell volume
39
(PCV), respectively. Adult GIN were recovered from the abomasum and small intestines
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2
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of all goats at the end of the experiment for counting and speciation. The CP levels were
41
highest for SL forage, intermediate for SL+BG, and lowest for BG forage samples, while
42
NDF and ADF values were the opposite, with highest levels in BG and lowest in SL
43
forage samples. Sericea lespedeza leaves had more CT than stems (16.0 vs 3.3 g/100 g
44
dry weight), a slightly higher percentage of PDs (98 vs 94%, respectively) and polymers
45
of larger mean degrees of polymerization (42 vs 18, respectively).
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There were no differences in average daily gain or blood PCV between the
47
treatment groups, but SL goats had lower FEC (P < 0.05) than the BG or SL+BG forage
48
goats throughout most of the trial. The SL+BG goats had lower FEC than the BG forage
49
animals by the end of the trial (week 8, P < 0.05). The SL goats had lower numbers (P <
50
0.05) of male H. contortus and tended to have fewer female (P <0.10) and total (P <
51
0.07) H. contortus compared with the BG goats. The predominant GIN in all the goats
52
was Trichostrongylus colubriformis (73% of total GIN).
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As a low-input forage with activity against pathogenic GIN (H. contortus), SL has
54
a potential to reduce producers’ dependence upon synthetic anthelmintics and also to fill
55
the autumn ‘window’ in good-quality fresh forages for goat grazing in the southern USA.
56 57 58
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1. Introduction
59
Gastrointestinal nematode (GIN) infection is a major hindering factor in small
60
ruminant production all over the world. Increased mortality and poor weight gains
61
contribute to the majority of losses in the sheep and goat industry (Wanyangu and Bain,
62
1994; Gatongi et al, 1997; Sharkhuu, 2001; Faye et al., 2003). Goats are even more 3
Page 3 of 29
susceptible than sheep to nematode infection (Tembely and Hansen, 1996). Goat
64
production is an important industry in the southeastern USA because of availability of
65
forages and also rising market demand, particularly for organically-produced meat
66
(Robinson, 2004). However, GIN control, particularly of Haemonchus contortus, a
67
pathogenic blood-feeder, is challenging in this region because the sub-tropical climate is
68
ideal for growth of the free-living stages of GIN (infective L3 larvae) on pasture. This
69
also applies to more northern parts of the USA that have periods of warm and humid
70
weather, such as during the summer months. Infection with Trichostongylus
71
colubriformis, and Teladorsagia circumcincta are also common in small ruminants
72
during cooler periods of the year and can lead to production losses (Miller, 1996).
73
Overuse of anthelminthic drugs to control small ruminant GIN has led to greatly
74
increased incidence of anthelmintic resistance world-wide, making this an unreliable
75
method to control parasites (Kaplan, 2004). Therefore, it is necessary to develop farm
76
management systems that can maintain high levels of animal production with reduced
77
reliance on anthelmintic intervention (Terrill et al., 2001; Shaik et al., 2006). There have
78
been a number of reports on alternative (non-synthetic) methods to control parasites in
79
small and large ruminants (Larsen, 1999; Kabagambe et al., 2000; Burke et al., 2004). A
80
plant-based control strategy with great potential for on-farm application, particularly for
81
small and limited resource producers, is feeding of forages containing condensed tannins
82
(CT; Niezen et al., 2002; Shaik et al., 2006).
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83
Niezen et al. (1995) reported lower fecal egg counts (FEC) and worm burdens (T.
84
colubriformis, T. circumcincta) in lambs grazing sulla (Hedysarum coronarium), a CT-
85
containing legume, compared with alfalfa (Medicago sativa), which contains no CT. 4
Page 4 of 29
Other CT-containing plant species with reported efficacy against GIN (H. contortus, T.
87
colubriformis, T. circumcincta) include big trefoil (Lotus pedunculatus; Niezen et al.,
88
1998), birdsfoot trefoil (L. corniculatus; Heckendorn et al., 2007), and sainfoin
89
(Onobrychus viciifolia; Heckendorn et al., 2006). In short-term studies, Min et al (2004;
90
2005) reported reduced FEC in goats grazing sericea lespedeza (SL; Lespedeza cuneata)
91
compared to a non-CT forage during the late spring-early summer period, while Shaik et
92
al (2006) reported reduced FEC and lower numbers of adult GIN (H. contortus, T.
93
colubriformis, T. circumcincta) in goats fed hay of SL compared with non-CT hay
94
(bermudagrass - BG; Cynodon dactylon).
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Sericea lespedeza is a widely adapted, perennial warm-season legume that grows
96
well on acidic and droughty soils in Georgia, USA (Hoveland et al., 1990). As a low-
97
input forage, SL is an economical feed resource in the southeastern USA during the
98
warmer (late spring/summer/early autumn) months, with similar animal performance as
99
perennial warm-season grasses, like BG (Ball et al., 2002). Several studies have reported
100
good efficacy of dried SL (hay, pellets) against GIN infection in goats during the late
101
summer-autumn period in the southern United States (Shaik et al., 2004; Terrill et al.,
102
2009; Gujja et al., 2013), but there have no reports on the potential anthelmintic effect of
103
goats consuming fresh (grazed) SL during this period. The objective of this study was to
104
determine the anthelminthic efficacy of fresh SL or perennial grass forage in the diet of
105
naturally-infected goats during the late summer-early autumn period in Georgia.
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2. Materials and methods
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A trial with intact male Spanish kids was completed at the Fort Valley State
110
University (FVSU) Agricultural Research Station, Fort Valley, GA during September,
111
October, and November, 2011. All husbandry practices and experimental procedures
112
used in the study were approved by the FVSU Animal Care and Use Committee.
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2.1. Forage paddocks
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Three paddocks (0.40 ha) were laid out in an area that had no previous grazing
118
animals for over 12 months using a completely randomized design for three treatments:
119
1) fresh SL forage (SL), 2) fresh grass (mixture of warm-season perennial species,
120
predominantly BG) forage (BG), and 3) a combination of SL and BG forage (SL+BG).
121
Spring and early summer growth on each of the paddocks was removed prior to the start
122
of the trial, and grazing was initiated on late summer, autumn regrowth. The SL and
123
SL+BG paddocks were fenced using electric netting (Electronet, Premier1 Supplies,
124
Washington, IA) with a solar charger. The BG forage paddock had permanent fencing.
125
Animals were set-stocked on each paddock throughout the study. To assure adequate
126
nutrition, all animals were given supplemental pelleted feed (Mossey Creek feed,
127
guaranteed analysis 14% crude protein, 4% crude fat, and 10% crude fiber; Mid-Georgia
128
Farm Supply, Montezuma, GA) at 0.45 kg/head/d for the initial 4 weeks and 0.23
129
kg/head/d for the remaining 4 weeks of the trial. The animals consumed all supplemental
130
feed offered during the trial, and there was adequate forage leaf material available
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Page 6 of 29
131
throughout the 8-week trial period in each paddock. All animals were allowed ad libitum
132
access to water throughout the study period.
133
2.2. Animals
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Thirty intact male Spanish kids (9 months old, 20.7 ± 1.1 kg) were used for this
137
experiment. The goats were allowed to acquire a natural infection by grazing infected
138
grass pasture for 4 weeks prior to the start of the trial. Animals were stratified by fecal
139
egg count (FEC) and then randomly assigned to forage paddocks such that each of the
140
goat groups (n = 10/paddock) had similar mean and standard deviation for total FEC.
141
After initiation of the trial, blood and fecal samples were taken weekly from individual
142
animals for FEC and packed cell volume (PCV) determination, respectively. Animal
143
body weights (BW) were taken at the start and end of the trial. At the end of 8 weeks, the
144
animals were transferred to the Fort Valley State University Meat Technology Center for
145
slaughter, blood and fecal samples were taken, and GIN from the abomasum and small
146
intestines were recovered for counting and speciation.
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2.3. Sampling and techniques
149
2.3.1. Forages
150
For forage quality determination, 10 random samples were taken from each
151
paddock prior to starting the trial using a 30.48 cm2 quadrat, with all plant material within
152
the quadrat cut to a 2.54 cm stubble. The samples from each paddock were composited,
153
dried at 60º C for 48 hours, weighed, and then ground for quality analyses. Subsamples of 7
Page 7 of 29
the supplemental commercial pellets were collected for processing and analysis as well.
155
A separate sample of SL was collected, immediately frozen, and then freeze-dried for CT
156
analysis. A portion of the freeze-dried whole plant material was hand separated into
157
leaves and stems, with leaf, stem, and whole plant samples ground for analysis.
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2.3.2. Forage sample analyses
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All oven-dried forage and feed pellet samples were ground to 1 mm particle size
161
using a Cyclotec grinder (Foss, Eden Prairie, MN) and then analyzed for dry matter
162
(DM), nitrogen (N), neutral detergent fiber (NDF), and acid detergent fiber (ADF).
163
Analysis of N was completed using a Carbon/Nitrogen analyzer (Vario Max, Elementar
164
Americas Inc., Mt. Laurel, NJ), with crude protein (CP) calculated as N x 6.25. Fiber
165
analyses (NDF and ADF) were completed using the method of Van Soest et al (1991)
166
using an ANKOM200/220 Fiber Analyzer (ANKOM Technology, Macedon, NY). Dry
167
matter was determined using AOAC (1984) protocols. All forage quality data are
168
presented on a DM basis.
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Leaf, stem, and whole plant samples of SL were freeze-dried and analyzed
170
directly for CT content and composition using the benzyl mercaptan thiolysis method
171
(Gea et al., 2011).
172 173
2.3.3. Chemicals and standards
174
Hydrochloric acid (36%), acetone (Analytical Reagent grade), dichloromethane
175
(HPLC grade), and methanol (HPLC grade) were obtained from ThermoFisher Scientific 8
Page 8 of 29
(Loughborough, U.K.). (±)-Dihydroquercetin (98%) was obtained from Apin Chemicals
177
(Abingdon, U.K.). Benzyl mercaptan (BM; 98%), (+)-catechin (C), (-)-epicatechin (EC),
178
(-)-gallocatechin (GC), (-)-epigallocatechin (EGC) were obtained from Sigma-Aldrich
179
(Poole, U.K.). Sephadex LH-20 was obtained from GE Healthcare (Little Chalfont, U.K.)
180
and C18 mini-cartridges from Agilent Technologies (Wokingham, U.K.). Deionized and
181
distilled water was obtained from a Milli-Q system (Millipore, Watford, U.K.).
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2.3.4. In situ thiolysis of condensed tannins
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Analysis of CT was performed according to a slightly modified method of Gea et
185
al, (2011). Freeze-dried material of whole SL plants, leaves and stems (200 mg) was
186
reacted with the thiolysis reagent (2 mL methanol, 1 mL of 3.3% HCl in methanol, and
187
100 μL BM) as previously described. Methanol (1 mL) was added to the mixture. The
188
sample was centrifuged at 4000 rpm for 3 min, and supernatant (1 mL) was transferred
189
into another screw cap glass tube. Distilled water (9 mL) was added to this supernatant.
190
After thorough mixing, the solution was added to a prepacked C18-mini-cartridge and
191
conditioned as in Gea et al. (2011). Methanol (2.5 mL) was added to elute thiolysis
192
reaction products under gravity and the internal standard, dihydroquercetin in methanol
193
(500 μL; 0.1 mg/ml), was added into the collection tubes. The reaction products were
194
analyzed by liquid chromatography-mass spectrometry (LC-MS; see below).
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195
In addition, free flavanol monomers were analyzed by LC-MS directly in plant
196
samples using the above ‘thiolysis reagent’, where the HCl-methanol (1 mL) and BM
197
(100 μL) reagents were replaced with methanol (1100 μL). 9
Page 9 of 29
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2.3.8. Analysis of thiolysis reaction products by liquid chromatography-mass
200
spectrometry (LC-MS)
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Flavan-3-ols and their BM adducts were identified by LC-MS analysis on a HPLC
202
Agilent 1100 series system and API-ES instrument Hewlett Packard 1100 MSD Series
203
(Agilent Technologies, Waldbronn, Germany). Samples (20 μL) were injected into a
204
HPLC system connected to an ACE C18 column (3 μm; 250 x 4.6 mm; Hichrom Ltd;
205
Theale; U.K.) fitted with a guard column at room temperature. The HPLC system
206
consisted of a G1379A degasser, G1312A binary pump, a G1313A ALS autoinjector, a
207
G1314A VWD UV detector, a G1316A column oven, and a personal computer with
208
LC/MSD Agilent ChemStation version A 10.01 software. The flow rate was 0.75 mL/min
209
using 1% acetic acid in water (solvent A) and HPLC-grade methanol (solvent B). The
210
following gradient program was employed: 0-52 min, 36% B; 52-60 min, 36-50% B
211
linear; 60-65 min, 50-100% B linear; 65-73 min, 100-0% B; 73-80 min, 0% B.
212
Chromatograms were recorded at 280 nm, and MS spectra were recorded in the negative
213
ionization scan mode between m/z 100 and 1000 using the following conditions: capillary
214
voltage, 3000 V; nebulizer gas pressure, 35 psig; drying gas, 12 mL/min; and dry heater
215
temperature, 350 °C. The concentrations of free flavanols were subtracted from
216
concentrations of terminal flavanols, which were released during thiolysis.
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217
This provided information on the tannin composition in terms of % terminal and
218
% extension flavanol units; it also allowed calculation of mean degree of polymerization
219
(mDP), % procyanidins (PC) and prodelphinidins (PD), and % cis- and trans flavanols 10
Page 10 of 29
220
according to Gea et al. (2011).
221
2.4. Animal samples
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2.4.1. Blood samples
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Blood samples were collected via jugular venipuncture into 3 mL vacutainer
226
blood tubes containing K2EDTA. Samples were returned to the lab for immediate
227
determination of packed cell volume (PCV) in duplicate using a microhematocrit
228
centrifuge and reader. Micro-hematocrit capillary tubes were filled with whole blood,
229
sealed, and then centrifuged for 10 minutes at 9245 x g in a hematocrit centrifuge (IEC
230
Model Mb, Fisher Scientific). The hematocrit values were read directly from the
231
centrifuge scale.
234
2.4.2. Fecal samples
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Fecal samples were collected directly from rectum of all animals weekly during
235
the trial to determine FEC using the modified McMaster technique (Whitlock, 1948). The
236
results were expressed as eggs per gram of feces (EPG) by the following formula:
237
Average number of eggs in 2 counting chambers x 100.
238 239 240 241
2.4.2. Recovery and counting of adult nematodes At the end of the trial, the goats were slaughtered at the USDA-approved abattoir at the Fort Valley State University Agricultural Research Station.
Fecal and blood
11
Page 11 of 29
samples were taken from individual animals for FEC and PCV determination,
243
respectively, and adult worms were recovered from the abomasum and small intestines as
244
described by Shaik et al (2006). After slaughter, the abomasum and small intestine of
245
each goat was ligated, opened, and the contents washed into plastic buckets. The contents
246
were brought up to 3 L with tap water, thoroughly mixed, and then two 5% aliquots (150
247
mL) taken into 250 mL storage containers. Approximately 100 mL of formalin (10%)
248
were added to each aliquot as a preservative for both abomasal and small intestinal
249
samples. These were brought up to 3 L and subsampled and preserved as described
250
previously. For processing, the adult worms in both (abomasal and small intestinal)
251
aliquots were washed on a mesh screen (53 microns) with tap water, the formalin
252
discarded, and the nematodes recovered into a 50 mL centrifuge tube. All the nematodes
253
in the tube were then counted and identified to species and sex using a Leica Zoom 2000
254
phase contrast microscope (Leica Microsystems Inc., Chicago, IL).
257
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2.5. Statistical analyses
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Fecal egg count and blood PCV data were analyzed using repeated measures
258
analysis as a completely randomized design (SAS, 2008). The model included treatment,
259
day as a repeated measure, and the interaction. When treatment effects were different at
260
P < 0.05, means were separated using LSD test. Adult GIN, beginning and ending BW,
261
and forage quality data were analyzed as a completely randomized design using the GLM
262
procedure of SAS. The FEC and adult GIN data were log-transformed (log FEC + 1)
263
prior to statistical analysis. Fecal egg count and adult GIN data were reported as least
264
squares means, with statistical inferences based upon log-transformed data analysis. 12
Page 12 of 29
265
Condensed tannin analyses were only completed for SL samples, so these data were not
266
subjected to statistical analysis.
3. Results
269 270
3.1. Chemical composition of pasture and pellet samples
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The CP, NDF, and ADF values for all forage samples are shown in Table 1. The
273
CP values were highest for the SL, intermediate for SL+BG, and lowest for the BG
274
forage, while the fiber values were the opposite, with lowest and highest values for the
275
SL and BG forages, respectively. The commercial pellet had 19.6, 15.0, and 9.4% CP,
276
NDF, and ADF, respectively.
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Leaves of SL had much higher tannin content than stems (16.0 vs 3.3 g/100 g dry
278
weight) (Table 2). Leaf CT were also much larger than stem CT, with mDP being 42 for
279
leaves and 18 for stems. Both CT are almost pure PD: i.e. 98% of CT are PDs in leaves
280
compared to 94% in stems. Both leaves and stems had small amounts of PC. The cis-
281
flavanol proportion is slightly higher in leaves (91%) than stems (84%). Whole plant
282
samples were intermediate in total CT content, mDP, PC, PD, and proportion of cis-
283
flavanols (Table 2).
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284
SL samples contained a small amount of free or non-tannin epigallocatechin
285
(0.2%) and 0.01% of gallocatechin (Table 3). The flavanols in terminal and extension
286
units were mostly epigallocatechin (87.4%), followed by gallocatechin (9.6%) and
287
epicatechin (2.6%). Catechin was only detected in extension units (0.5%). 13
Page 13 of 29
288 289
3.2. Fecal egg count Fecal egg counts were similar between the three treatment groups at the start of
291
the trial (week 0). Two weeks after initiation of the trial, FEC from goats consuming SL
292
forage were lower (P < 0.04) than for animals grazing the other two pastures (BG and
293
SL+BG) (Figure 1). At the 3rd and 4th week samplings, FEC results were not different
294
among treatment groups, but starting from the 5th week through the end of the trial, FEC
295
of the SL goats were significantly lower (P < 0.02) compared with the other two
296
treatment groups. Compared to control goats, the SL animals had 82.4% lower FEC by
297
week five, and this increased to 95.4% reduction by the end of the trial. The FEC values
298
for the SL+BG treatment goats were not different from controls until the final week of
299
the trial, when the SL+BG group FEC were 71.5% lower (P < 0.03).
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302
3.3. Packed cell volume (PCV)
The PCV values for all the goats were initially low (ranged from 14-17%) and
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303
increased throughout the trial (final sampling values ranged from 27-28%). There was no
304
significant difference (P = 0.59) among the three treatment groups throughout the
305
experiment (Figure 2).
306 307
3.4. Body weights
308
Initial and final body weights and ADG for all the goats are presented in Table 4.
309
There was no effect of pasture forage type on final live weight or gain per day in the
310
goats. 14
Page 14 of 29
311 312
3.5. Adult nematodes The adult nematode worm species recovered from abomasal samples were H.
314
contortus and T. circumcincta. Total GIN and number of males and females counted for
315
each nematode species are presented in Table 5. Number of adult H. contortus males was
316
lower (P < 0.05) in goats consuming SL pasture forage compared with BG pasture
317
animals, and female and total H. contortus tended to be lower (P < 0.09, P < 0.07,
318
respectively) in the SL groups compared with BG goats. Although there was no
319
significant difference in small intestinal nematodes (T. colubriformis) between the
320
groups, total and male T. colubriformis numbers tended to be lower (P < 0.11, P < 0.06,
321
respectively) in the SL compared with the mixed forage (BG+SL) goats. Overall, there
322
was a trend (P < 0.11) for lower total worms in the SL group compared with the SL+BG
323
goats.
324
326 327 328
4. Discussion
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4.1. Condensed tannins in sericea lespedeza Sericea lespedeza has a surprisingly high CT content (12.5 g/100 g) and a
329
relatively rare CT composition (Table 2). SL tannins were almost pure PDs (97%) of high
330
molecular weight (mDP = 33, which corresponds to 10,100 Daltons) and also had a high
331
proportion of cis-flavanols (90%), which is due to a preponderance of epigallocatechin
332
(87%) and gallocatechin (10%) units (Table 3). The same tannin method has previously
333
been used to screen sainfoin accessions (Stringano et al., 2012). This germplasm 15
Page 15 of 29
collection covered CT contents ranging from 0.6 to just 2.8 g/100 g dry weight. Only 16
335
out of 37 accessions had tannins with more than 80% PDs, just 1 accession had more than
336
90% PDs and 15 accessions had polymers with mDP-values above 30. The finding that
337
leaves not only had a higher CT concentration than stems, but also a higher PD
338
percentage and larger CT polymers (mDP-value) agrees with results from sainfoin
339
(Theodoridou et al., 2010 and 2011).
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341
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4.2. Anti-parasitic effects
The most effective treatment in this study against GIN infection in goats,
343
particularly for H. contortus, was fresh SL forage, which confirms previous reports on the
344
anthelmintic properties of this plant in fresh and dried forms for small ruminants (Min et
345
al., 2004; 2005; Shaik et al., 2006; Terrill et al., 2007; 2009). Consumption of mixed
346
SL+BG fresh forages also had some effect in reducing GIN infection in goats compared
347
with grass (BG) only, but the effect took longer (Figure 1), similar to results of Burke et
348
al (2012a) with lambs grazing mixed grass and SL.
350 351
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4.2.1. Fecal egg counts
The initial reduction (P < 0.05) in FEC in goats consuming fresh SL compared
352
with grass (BG) (33% reduction) or SL+BG forage (56%) occurred two weeks after the
353
animals were turned onto the pastures, and these differences increased throughout the
354
trial, to 95 and 84%, respectively, by the final sample date. The goats consuming SL+BG
355
forage had 41% lower FEC than control (BG) animals by week 5 of the study, and these
356
differences also increased throughout the remainder of the trial, but reached significance 16
Page 16 of 29
(P < 0.05) only at the final sample date (71% reduction). The FEC reductions attained by
358
consuming fresh SL alone or in mixture with BG in autumn confirm past reports for goats
359
grazing SL in summer (Min and Hart, 2003; Min et al., 2005) or fed SL in dried forms
360
(Shaik et al., 2006; Terrill et al., 2009). In contrast, Burke et al. (2012a) reported no
361
effect of summer-grazed SL on predominantly Trichostrongylus spp. infected goats, but
362
reduced (P < 0.05) FEC in lambs (predominantly H. contortus in control lambs) grazing
363
SL alone or in a mixture with BG compared with grass pastures only (Burke et al.,
364
2012b). The delay in FEC reduction for the goats consuming fresh SL+BG forage
365
compared to BG alone in the current investigation may be due to several factors. Based
366
upon daily observation, the goats in the mixed SL+BG pasture initially grazed the non-
367
SL components for the first 2-3 weeks, after which they grazed the BG and SL more
368
evenly. In the SL paddock (which also had a small amount of grasses along the fence
369
lines), the goats started grazing the SL plants within 2-3 days. In previous reports,
370
reduction in FEC due to SL feeding was slower during times of the year when H.
371
contortus was not the primary GIN, such as in the autumn or winter months (Shaik et al.,
372
2004; Terrill et al., 2009). The current study was completed during mid-September to
373
mid-November with goats that had a non-H. contortus-dominant infection (73% T.
374
colubriformis). This may also explain the lack of a treatment effect on PCV values in the
375
animals, as anemia scores have been reported to be less affected by SL when H. contortus
376
is not the predominant GIN (Terrill et al., 2009). However, overall low numbers of adult
377
H. contortus in all the animals is more likely the reason for the lack of difference in
378
anemia scores between treatment groups (Figure 2). In the current study, differences in
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379
GIN infection or FEC reduction could be due to the higher quality of SL relative to BG,
380
although forage quality was only measured initially. Regardless of the type of GIN infection, a potential long-term benefit of reduced
382
fecal egg shedding in goats consuming fresh SL forage in autumn may be reduced
383
subsequent pasture infectivity (number of GIN L3 per unit pasture DM), such as in the
384
following spring.
cr
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381
386
us
385
4.2.2. Adult nematodes
The goats consuming fresh SL forage had 48% lower numbers of adult H.
388
contortus in the abomasum (P < 0.07) compared with control animals, with greater
389
differences in males (P < 0.05) than in females (P < 0.10). These results contrast with
390
those of Shaik et al. (2006), who reported a greater reduction effect of SL hay on female
391
than male GIN, including H. contortus. Reasons for the differential sex effect in the
392
current study are unclear, but the most important effect from the standpoint of reducing
393
egg production is obviously with the females. The SL goats also had 34% fewer numbers
394
of T. circumcincta than controls, and 44% fewer total abomasal (H. contortus + T.
395
circumcincta) than small intestinal worms, but the differences were not significant (Table
396
5). There have been few reports of effects of grazed SL on worm burdens in small
397
ruminants, but reports from sheep and goat feeding trials with dried SL (ground and
398
unground hay, pellets) have found greater effects on H. contortus adults than for other
399
GIN species (Shaik et al., 2006; Lange et al., 2006; Terrill et al., 2007; 2009). Shaik et al
400
(2006) reported reductions of 70, 26, and 40% for adult H. contortus, T. circumcincta and
401
T. colubriformis, respectively, in animals fed SL long hay as opposed to BG hay as 75%
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of their ration. In the current study, initially goats would be expected to receive
403
approximately 30% of their diet from forage if consuming 3% of BW. Later, this
404
increased to about 60% on those same principles (CP and energy would have been
405
greatest for goats consuming SL). Regardless, the overall reduction effect of SL on adult
406
worms was against abomasal as opposed to small intestinal GIN.
ip t
402
Sericea lespedeza has been reported to be an effective bioactive forage against
408
small ruminant GIN when used in fresh (grazed) or dried forms (Min et al., 2005; Shaik
409
et al., 2006). These effects have been attributed to both direct effects on the adult worm
410
(killing the worm or reducing female fecundity; Min et al., 2005; Shaik et al., 2006) or
411
indirect effects of improving the nutritional status of the host by protecting protein from
412
rumen degradation and increasing amino acid flow to the small intestines (Waghorn et
413
al., 1997), allowing increased resilience to GIN infection (Moore et al., 2008). Definitive
414
differences between direct and indirect effects of SL on GIN (CT versus increased
415
protein effect) could not be separated because of the experimental design in the current
416
investigation. There is evidence for direct effects of fresh SL on GIN in the current
417
investigation (reduced FEC and H. contortus numbers), but there were no treatment
418
differences in animal performance (weight gains). All animals gained from 80 to 110 g/d
419
throughout the trial, suggesting that the goats all had adequate nutrition, which allowed
420
them to continue to be moderately productive despite differences in GIN status.
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421 422
5. Conclusions
423
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Autumn consumption of fresh SL forage reduced FEC and H. contortus numbers
425
in goats compared with perennial warm-season grass (BG) forage, while giving
426
comparable animal performance. It is possible that SL is particularly effective at
427
controlling H. contortus due to its unusual tannins, which are almost pure prodelphinidins
428
of relatively high molecular weight. A long-term benefit of autumn grazing of SL pasture
429
may be reduced subsequent pasture infectivity due to lower numbers of GIN eggs on
430
pasture. As a low-input crop, and with its potential use as a natural anti-parasitic agent,
431
SL is an economical forage for small ruminant production in the southern USA, while
432
potentially reducing farmers’ dependence upon synthetic anthelmintics.
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Acknowledgements
This research was supported by the USDA NIFA Organic Research and
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Education Initiative (Project no. 2010-51300-21641). A. Ramsay acknowledges financial
437
support from the European Union (PITN-GA-2011-289377, ‘LegumePlus’).
439 440 441 442 443 444 445 446 447 448 449 450 451 452
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References
AOAC, 1984. Official Methods of Analysis (14th Ed.). Association of Official Analytical Chemists, Washington, D.C. Ball, D.M., Hoveland, C.S., Lacefield, G.D., 2002. Southern Forages, Third edition, Graphic Communications Corporation, Lawrenceville, GA, 322p. Burke, J.M., Miller, J.E., Mosjidis, J.A., Terrill, T.H., 2012a. Use of a mixed sericea lespedeza and grass pasture system for control of gastrointestinal nematodes in lambs and kids. Vet. Parasitol. 186, 328-336. Burke, J.M., Miller, J.E., Mosjidis, J.A., Terrill, T.H., 2012b. Grazing sericea lespedeza for control of gastrointestinal nematodes in lambs. Vet. Parasitol. 186, 507-512. Burke, J.M., Miller, J.E., Olcott, D.D, Olcott, B.M, Terrill, T.H., 2004. Effect of copper oxide wire particles dosage and feed supplement level on Haemonchus contortus infection in lambs. Vet. Parasitol. 123, 235-243. 20
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Faye, D., Leak, S., Nouala, S., Fall, A., Losson, B., Geerts, S., 2003. Effects of gastrointestinal helminth infections and plane of nutrition on the health and productivity of F1 (West African Dwarf × Sahelian) goat crosses in The Gambia. Small Rumin. Res. 50, 153-161. Gatongi, P.M., Scott, M.E., Ranjan, S., Gathuma, J.M., Munyua, W.K., Cheruiyot, H., Prichard, R.K., 1997. Effects of three nematode anthelmintic treatment regimes on flock performance of sheep and goats under extensive management in semiarid Kenya. Vet. Parasitol. 68, 323-336. Gea, A., Stringano, E., Brown, R.H., Mueller-Harvey, I., 2011. In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high throughput germplasm screening. J. Agric. Food Chem. 59, 495-503. Gujja, S., Terrill, T.H., J.A. Mosjidis, J.E. Miller, A. Mechineni, D.S. Kommuru, S.A. Shaik, B.D. Lambert, N.M. Cherry, and J.M. Burke. 2013. Effect of supplemental sericea lespedeza leaf meal pellets on gastrointestinal nematode infection in grazing goats. Vet. Parasitol. 191, 51-58.
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Heckendorn F., Haring, D.A., Maurer, V., Zinsstag, J., Lanfhans, W., Hertzberg, H. 2006. Effect of sainfoin (Onobrychis viciifolia) silage and hay on established populations of Haemonchus contortus and Cooperia curticei in lambs. Vet. Parasitol. 142, 293-300. Heckendorn F., Haring, D.A., Maurer, V., Senn, M., Hertzberg, H. 2007. Individual administration of three tanniferous forage plants artificially infected with Haemonchus contortus and Cooperia curticei. Vet. Parasitol. 146, 123-134. Hoveland, C.S., Windham, W.R., Boggs, D.L., Durham, R.G., Calvert, G.V., Newsome, J.F., Dobson, Jr., J.W., Owsley, M., 1990. Sericea production in Georgia. Research Bulletin 393; The Georgia Agricultural experiment stations, College of Agriculture, The University of Georgia. Kabagambe, E.K., Barras, S.R., Li., Y., Pena, M.T., Smith, W.D., Miller, J.E., 2000. Attempts to control haemonchosis in grazing ewes by vaccination with gut membrane proteins of the parasite. Vet. Parasitol. 92, 15-23. Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol. 20, 477-481. Lange, K., Olcott, D.D., Miller, J.E., Mosjidis, J.A., Terrill, T.H., Burke, J.M., Kearney, M.T., 2006. Effect of sericea lespedeza (Lespedeza cuneata) fed as hay, on natural and experimental Haemonchus contortus infections in lambs. Vet. Parasitol. 141, 273-278. Larsen, M., 1999. Biological control of helminths. Int. J. Parasitol. 29, 139-146. Miller, J.E. 1996. Controlling goat parasites in the southeast. pp. 80-82 In: Proc. Southeast Reg. Meat Goat Prod. Sym., 21-24 February, 1996, Florida A&M University, Tallahassee, FL. Min, B.R., Hart, S.P., 2003. Tannins for suppression of internal parasites. J. Anim. Sci. 81 (E-Suppl. 2), 102-109. Min, B.R., Pomroy, W.E., Hart, S.P., Sahlu, T., 2004. The effect of short-term consumption of a forage containing condensed tannins on gastro-intestinal nematode parasite infections in grazing wether goats. Small Rumin. Res. 51, 279283.
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Min, B.R., Hart, S.P., Miller, D., Tomita, G.M., Loetz, E, Sahlu, T., 2005. The effect of grazing forage containing condensed tannins on gastro-intestinal parasite infection and milk composition in Angora does. Vet. Parasitol. 130, 105-113. Moore, D.A., Terrill, T.H., Kouakou, B., Shaik, S.A., Mosjidis, J.A., Miller, J.E., Vanguru, M., Kannan, G., Burke, J.M., 2008. The effects of feeding sericea lespedeza hay on growth rate of goats naturally infected with gastrointestinal nematodes. J. Anim. Sci. 86, 2328-2337. Niezen, J.H., Waghorn, T.S., Charleston, W.A.G., Waghorn, G.C., 1995. Growth and gastrointestinal nematode parasitism in lambs grazing either lucerne (Medicago sativa) or sulla (Hedysarum coronarium) which contains condensed tannins. J. Agric. Sci. 125, 281-289. Niezen, J. H., Robertson, H.A., Waghorn, G.C., Charleston, W.A.G., 1998. Production, fecal egg counts and worm burdens of ewe lambs which grazed six contrasting forages. Vet. Parasitol. 80, 15–27. Niezen, J.H., Charleston, W.A.G., Robertson, H.A., Shelton, D., Waghorn, G.C., Green, R., 2002. The effect of feeding sulla (Hedysarum coronarium) or Lucerne (Medicago sativa) on lamb parasite burdens and development of immunity to gastrointestinal nematodes. Vet. Parasitol. 105, 229-245. Robinson, J., 2004. Pasture Perfect: The Far-reaching Benefits of Choosing Meat, Eggs, and Dairy Products from Grass-fed Animals. Vashon Island Press, Vashon, WA, 160 pp. SAS Institute, 2008. SAS/STAT Software: changes and enhancements. Release 9.2, SAS Technical Report, SAS Institute, Cary, NC. Shaik, S.A., Terrill, T.H., Miller, J.E., Kouakou, B., Kannan, G., Kallu, R.K., Mosjidis, J.A., 2004. Effects of feeding sericea lespedeza hay to goats infected with Haemonchus contortus. SAJ Anim. Sci. 34, 234-236. Shaik, S.A., Terrill, T.H., Miller, J.E., Kouakou, B., Kannan, G., Kaplan, R.M., Burke, J.M., Mosjidis, J.A., 2006. Sericea lespedeza hay as a natural deworming agent against gastrointestinal nematode infection in goats. Vet. Parasitol. 139, 150-157. Sharkhuu, T., 2001. Helminths of goats in Mongolia. Vet. Parasitol. 101, 161-169. Stringano, E., Hayot Carbonero, C., Smith, L.M.J., Brown, R.H., Mueller-Harvey, I., 2012. Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection. Phytochem. 77, 197-208. Tembely, S., Hansen, J.W., 1996. Helminth Diseases of Small Ruminants in the Tropics: A Review of Epidemiology and Control Strategies. 22-25 April 1996; Bogor, Indonesia, 123-127. Terrill, T.H., Kaplan, R.M., Larsen, M., Samples, O.M., Miller, J.E., Gelaye, S., 2001. Anthelmintic resistance on goat farms in Georgia: efficacy of anthelmintics against gastrointestinal nematodes in two selected goat herds. Vet. Parasitol. 97, 261-268. Terrill, T.H., Mosjidis, J.A., Moore, D.A., Shaik, S.A., Miller, J.E., Burke J.M., Muir, J.P., Wolfe, R., 2007. Effect of pelleting on efficacy of sericea lespedeza hay as a natural dewormer in goats. Vet. Parasitol. 146, 117-122.
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Terrill, T.H., Dykes, G.S., Shaik, S.A., Miller, J.E., Kouakou, B., Kannan, G., Burke, J.M., Mosjidis, J.A., 2009. Efficacy of sericea lespedeza hay as a natural dewormer in goats: Dose titration study. Vet. Parasitol. 163, 52-56. Theodoridou, K., Aufrère, J., Andueza, D., Pourrat, J., Le Morvan, A., Stringano, E., Mueller-Harvey, I., Baumont, R. 2010. Effects of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep. Anim. Feed Sci. Technol. 160, 23-38. Theodoridou, K., Aufrère, J., Andueza, D., Le Morvan, A., Picard, F., Stringano, E., Pourrat, J., Mueller-Harvey, I., Baumont, R., 2011. Effect of plant development during first and second growth cycle on chemical composition, condensed tannins and nutritive value of three sainfoin (Onobrychis viciifolia) varieties and lucerne. Grass For. Sci. 66, 402–414. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597. Waghorn, G.C., Ulyatt, M.J., John, A., Fisher, M.T., 1987. The effect of condensed tannins on the site of digestion of amino acids and other nutrients in sheep fed on lotus. Brit. J. Nutr. 57, 115–126. Wanyangu, S.W., Bain, R.K., 1994. The impact of helminths in small ruminant production in tropical Africa. Kenyan Vet. 18, 104-106. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg counting technique apparatus. J. Coun. Sci. Ind. Res. 21, 177-180.
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541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564
23
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568
Table 1. Chemical composition of fresh sericea lespedeza (SL), bermudagrass (BG), and
569
a mixture of SL+BG forage (All values expressed on dry matter basis). Constituent1
570
NDF %
BG
6.3
71.7
SL
14.7
35.4
SL+BG
10.8
56.0
1
ADF %
ip t
CP %
cr
35.2
us
Forage type
25.2 33.4
CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber.
an
571 572
Table 2. Analysis of tannin content (g/100 dry weight) and composition of sericea
574
lespedeza (SL) in terms of mean degree of polymerization (mDP), percentage of
575
prodelphinidins (PD) and procyanidins (PC), percentage of cis- and trans-flavanols (±
576
standard error).
d
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577
M
573
Plant material CT content
mDP
PD
PC
Cis
Trans
%
%
%
%
(SL)
(g/100g)
Whole plant
12.5±0.8
32.8±0.9 96.9±0.1 3.1±0.1 90.0±0.0 10.0±0.0
Leaves
16.0±1.8
41.9±1.8 98.0±0.1 2.0±0.1 90.8±0.0 9.17±0.0
Stems
3.33±0.3
17.8±0.8 93.6±0.1 6.4±0.1 83.8±0.2 16.2±0.2
578
24
Page 24 of 29
ip t cr
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Table 3. Flavanol composition of free monomers and tannins in the whole plant, in leaves and stems of sericea lespedeza (SL) (±
Plant material
Flavanol monomers
(SL)
% GC
% EGC
M an
standard error).
Terminal units % GC
% EGC
%C
% EC
Extension units (BM1-adducts) % GC-BM % EGC-BM
% C-BM
% EC-BM
0.01±0.004 0.19±0.02 0.40±0.03 2.20±0.10
ND
0.46±0.11
9.18±0.03
85.2±0.10
0.46±0.02
2.15±0.01
Leaves
0.02±0.005 0.22±0.02 0.36±0.02 1.88±0.14
ND
0.16±0.01
8.51±0.02
87.2±0.29
0.29±0.01
1.57±0.15
ND
0.49±0.02
14.9±0.22
73.6±0.03
0.75±0.05
5.20±0.03
ND
ND = none detected 1
0.13±0.02 0.61±0.02 4.54±0.24
ce pt
Stems
ed
Whole plant
Ac
BM = benzyl mercaptan; GC = gallocatechin; EGC = epigallocatechin, C = catechin; EC = epicatechin.
25
Page 25 of 29
ip t
Table 4. Body weights and average daily gain (ADG) in goats consuming fresh sericea lespedeza (SL), bermudagrass (BG), or a mixture of SL+BG forage (± standard error).
Forage type
cr
Body weight (kg)
Final live weight
ADG (g)
BG
21±1.2
26±1.8
85±14.1
SL
20±1.1
26±1.7
108±13.3
BG + SL
21±1.0
25±1.6
81±12.6
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Initial live weight
26
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Table 5. Total male and female nematodes (± standard error) recovered from abomasum and small intestines of goats consuming fresh sericea lespedeza (SL), bermudagrass
Adult nematodes
BG
SL
Abomasum (total)
1468±411
817±390
H. contortus
1206±381
627±362
898±362
Male
509±193b1
us
Pasture treatment
ip t
(BG), or a mixture of SL+BG forage.
309±183a
391±183ab
318±191
507±191
113±42
119±42
53±38
51±38
60±15
68±15
3594±505
4725±505
3974±532
3594±505
4725±505
1696±228
1604±217
2184±217
Female
2279±310
1988±295
2621±295
Total worms
5442±720
4411±683
5780±683
T. circumcincta
170±44
Male
116±40
Female
54±15
T. colubriformis
1
1055±390
cr
d
Ac ce p
Male
3974±532
te
Small intestine (total)
an
697±202
M
Female
BG + SL
Row means with differing superscripts differ at P < 0.05. If no superscripts, then not
different (P > 0.05).
27
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Figure 1. The effect of consuming fresh sericea lespedeza (SL), bermudagrass (BG) or
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parasitized goats.
d
SL+BG mix forage on gastrointestinal nematode eggs per gram (EPG) of feces from
28
Page 28 of 29
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Figure 2. The effect of consuming fresh sericea lespedeza (SL), bermudagrass (BG) or
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d
SL+BG mix forage on blood packed cell volume (PCV) of parasitized goats.
29
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