Tarleton State University, Department of Animal Science and Wildlife Management, Stephenville, TX 76402, USAAngelo State University, Agriculture Department, ASU Station #10888, San Angelo, TX 76909-0888, USATexas AgriLife Research, 7887 U.S. Hwy 87 N., San Angelo, TX 76901, USATexas AgriLife Research, 1229 U.S. Hwy 281 N., Stephenville, TX 76401, USATexas A&M University, College of Veterinary Medicine, Suite 101, College Station, TX 77843-4461, USA
a r t i c l e i n f o
rticle history:eceived 13 August 2012eceived in revised form 10 April 2013ccepted 13 April 2013
A modified larval migration inhibition assay was used to determine if redberry juniper(Juniperus pinchotii Sudw.) can reduce Haemonchus contortus in vitro motility and increaseivermectin (IVM) efficacy. Ruminal fluid was mixed with buffer solution and either nomaterial (CNTL) or Tifton 85 Bermudagrass hay (T85), dried juniper (DRY), fresh juniper(FRE), or distilled juniper terpenoid oil (OIL) to make treatment solutions and anaerobicallyincubated for 16 h. For Trial 1, larvae were incubated in CNTL, T85, DRY, or IVM. DuringTrial 2, larvae were incubated in CNTL, DRY, FRE, or OIL for 4 h. Trials 3 (CNTL or OIL) and4 (CNTL, DRY or FRE) evaluated larvae after incubation in treatment solution for 2 h, thenincubated an additional 2 h in various IVM doses (0, 0.1, 1, 3, and 6 �g/mL IVM) and placedonto a screen. Larvae that passed through the 20-�m screen within a 96-well plate wereconsidered motile. Larvae incubated in CNTL or T85 had similar (P = 0.12) motility, but larvaeincubated in DRY were less (P < 0.02) motile than larvae incubated in CNTL or T85 (Trial 1).During Trial 2, adding DRY, FRE, or OIL reduced (P < 0.001) larval motility as comparedto CNTL. A treatment × IVM dose interaction (P = 0.02) was observed during Trial 3, due
to OIL unexpectedly decreasing IMV efficacy at IVM concentrations of 1 (P = 0.07), 3, and 6(P < 0.002) �g/mL. No treatment × IVM dose interaction (P = 0.57) was observed during Trial4, but larvae incubated in DRY had less (P < 0.004) total motility than larvae incubated inCNTL or FRE. Juniper forage material reduced in vitro H. contortus larval motility, but IVMefficacy was increased only by initially incubating larvae in DRY.
. Introduction
Gastrointestinal parasite control programs based on
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ynthetic dewormers are failing because of widespreadewormer resistance (Craig and Miller, 1990; Prichard,001); thus, alternative strategies are necessary. Plants
and their extracts, e.g. condensed tannins (CT), can directlyor indirectly decrease larval motility (Athanasiadou et al.,2001; Min and Hart, 2003; Molan et al., 2004) and totalworm burden (Niezen et al., 1995), and essential oils canbe bactericidal (Chao et al., 2000) and toxic to nema-todes (Lei et al., 2010). Redberry juniper (Juniperus pinchotii
al., Effect of using redberry juniper (Juniperus pin-d increase ivermectin efficacy. Vet. Parasitol. (2013),
Sudw.) contains CT and terpenoids and is consumedby sheep and goats (Riddle et al., 1996; Whitney andMuir, 2010) but its effects on Haemonchus contortus areunknown.
Over 20 million acres of Texas rangelands are infestedwith juniper (Ansley et al., 1995) and managing itsencroachment by mechanical and chemical methods isnot always cost effective (Lee et al., 2001). Increasing the“value” of juniper as a biological anthelmintic could poten-tially increase mechanical harvesting of this invasive plant,thereby increasing top-growth biomass for livestock con-sumption, and reducing its encroachment. The objectivesof this study were to use a modified larvae migration inhibi-tion (LMI) assay to determine if redberry juniper can reduceH. contortus in vitro motility and increase IVM efficacy.
2. Materials and methods
2.1. Juniper forage, juniper terpenoid oil, and other testmaterials
Fresh juniper material (stems <3.6 cm and leaves) wasrandomly harvested, immediately placed on dry ice, andchopped into approximately 3-cm pieces; a subsample wasimmediately lyophilized, stored at −80 ◦C, and used in theLMI assays within 2 days of harvesting; other subsam-ples were collected and immediately transferred on dryice to determine terpenoid oil concentrations. To collectjuniper terpenoid oil, fresh juniper branches with leaveswere chopped, placed into water, and distilled.
The terpenoid oil (OIL) was stored in glass vials at−80 ◦C for less than 3 months. Total and individual ter-penoid concentrations were presumed stable accordingto previous reports related to storing juniper material(Utsumi et al., 2006; Adams, 2010). For dried juniper,randomly selected branches from multiple trees were col-lected, chopped, dried at 55 ◦C in a forced-air oven for96 h to reduce volatile terpenoid concentrations, and storedat −80 ◦C. The CT extract used in this study originatedfrom the Quebracho Colorado tree (Schinopsis lorentziiGriseb. Engl.; UNITAN S.A.I.C.A., Argentina), which con-tains approximately 73% CT and 19% simple phenolics andis 100% water soluble at 40–45 ◦C (Athanasiadou et al.,2001). Ivermectin (IVM, 0.08% solution; Ivomec, MerialLimited, Iselm, NJ) and dimethyl sulfoxide (DMSO; anhy-drous, 99.9%; Sigma–Aldrich, St. Louis, MO) were also used.
2.2. In vitro rumen system
In Trial 1, ruminal fluid was collected from a mature,fistulated goat (Boer × Spanish) consuming Tifton 85Bermudagrass hay. For Trials 2, 3, and 4, ruminal fluid wascollected from three fistulated goats fed alfalfa hay andpellets (2% of body weight; BW). Each trial was replicatedusing fresh ruminal fluid that was never collected morefrequently than once every 2 days. To dislodge some of theparticle-associated bacteria, approximately 150 g of rumenmaterial was also added to each pre-warmed thermos,which was vigorously shaken for 1 min. The ruminal fluidwas pooled, strained through four layers of cheesecloth,and flushed with CO2 to ensure an anaerobic environ-
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ment. Strained ruminal fluid was mixed with McDougal’sbuffer solution (McDougal, 1948) at a ratio of 1:4, butonly using 25% of suggested urea because nitrogen con-centration can affect H. contortus in vitro motility (Howell
PRESSsitology xxx (2013) xxx– xxx
et al., 1999) and nutrition and parasite interactions canoccur (Coop and Holmes, 1996). Containers were subse-quently flushed with CO2 whenever opened. Initial pH ofthe ruminal fluid–buffer mixture remained between 6.8and 6.9 as it was transferred to incubation jars and placedinto a DaisyII Incubator (Ankom Technology, Fairport, NY)for 16 h at 37 ◦C to allow the solution to equilibrate. Spe-cific details of the DaisyII incubator have been previouslyreported (Robinson et al., 1999).
Test material was then thoroughly mixed with the rumi-nal fluid–buffer mixture to make the treatment solutions,and amount of test material placed into the incubationjar represented a 55-kg sheep consuming 2.5% of BW(dry matter; DM basis) and the following were consid-ered: (1) juniper consumed at 40% of diet DM; (2) dry andfresh juniper contained 95% and 60% DM, respectively; (3)juniper material contained 4% terpenoid oil (DM basis);and (4) 1 mL of oil = 0.825 g of oil. A conversion factor of0.125 (1:8 ratio) was used to adjust the quantity of testmaterial placed into the jar, considering that the incubationjar was 1 L and assuming an 8-L rumen (Purser and Moir,1966). After incubating test material, ruminal fluid–buffersolution was centrifuged at 970 × g for 5 min to removedebris from buoyant top layer. Aliquots (5 mL) of the mid-dle supernatant layer were transferred to 2 specimen cups;1 cup was used to incubate ensheathed H. contortus larvaeand 1 was used for treatment solution only. Approximately1125 larvae (0.75 mL) were then added to the cups resultingin a final volume of 5.75 mL (approximately 195 larvae/mL).Cups were flushed with CO2, sealed, and incubated at 37 ◦Cwith treatment solution.
2.3. Laboratory analysis of juniper material and oil andrumen fluid
Nitrogen was analyzed by a standard combustionmethod (990.03; AOAC, 2006) and crude protein (CP) cal-culated as 6.25 × N. Neutral detergent fiber (NDF) andacid detergent fiber (ADF) were analyzed with Van Soestet al. (1991) procedures modified for an Ankom 2000 FiberAnalyzer (Ankom Technol. Corp., Fairport, NY) without cor-recting for residual ash, using �-amylase, and omittingsodium sulfite. Ash was analyzed by a standard method(942.05; AOAC, 2006). Part of each sample was dried in aforced-air oven at 103 ◦C for 3 h to determine DM concen-tration. Condensed tannins in freeze-dried, fresh redberryjuniper leaves were assayed for soluble, protein-bound,and fiber-bound fractions by methods described by Terrillet al. (1992). Samples were oven-dried and standards wereprepared from redberry juniper as recommended by Wolfeet al. (2008). Dry and fresh juniper were analyzed for per-centage of terpenoid oil concentration using proceduresdescribed by Tellez et al. (1997).
2.4. Preparation of L3 larvae for LMI assay
Larvae used in Trial 1 were copricultured from fecal
al., Effect of using redberry juniper (Juniperus pin-d increase ivermectin efficacy. Vet. Parasitol. (2013),
material collected from Spanish × Boer cross goats previ-ously infected with H. contortus. As previously described(Whitney et al., 2011), two strains of H. contortus IVM-resistant L3 larvae were copricultured from fecal material
ollected from pygmy–Boer cross goats and lambs for Tri-ls 2, 3, and 4. DrenchRite assays were used to confirmVM resistance in the goat larvae according to the direc-ions of the manufacturer DrenchRite assay (DrenchRitesers Guide, 1996, Horizon Technology, Australia) withodifications as described by Kaplan et al. (2007). The
VM resistance of larvae from the lambs were confirmedy using a FEC reduction test (Coles et al., 1992). Larvalotility was similar between both strains incubated in a
ontrol ruminal fluid–buffer solution and in various IVMoncentrations (evaluated across studies; data not shown).
Baermann apparatus (Dinaburg, 1942) was used to collectnfective L3 larvae, which were considered to be from IVM-esistant strains. Haemonchus contortus was the only larvaepecies identified. Approximately 1000–1500 larvae/mLater were stored (<2.5 months) horizontally in tissue cul-
ure flasks with canted necks and a vented caps (Nunclanell culture flask, Thermo Scientific, Rochester, NY) con-aining 3 mm of room-temperature water and 5 �g/mL ofungicide (Fungizone, 250 �g/mL stock solution; Omegacientific Inc., Torzana, CA, USA). During this study, per-entages of viable and ensheathed larvae stored in theasks remained greater than 92% and 99%, respectively.arvae were not artificially exsheathed because this studyttempted to represent a true in situ ruminal environment.
.5. Larval migration inhibition assay
The LMI assay used in the current study was similar torocedures reported by Whitney et al. (2011) who modifiedrocedures developed by Kotze et al. (2006) and Waglandt al. (1992) by using a 96-well MultiScreen filter plateMillipore Corp., Bedford, MA USA). The plate consists of
plastic underdrain with 96 tear-drop shaped wells and aemovable 20-�m screen plate, which has been reported tonsure active L3 migration while preventing L3 from fallinghrough the screen (Rabel et al., 1994). Wells of the plasticnderdrain were filled with 150 �L of treatment solution
mmediately prior to the screen being placed into the solu-ion, making sure no air bubbles were present.
After larvae incubated in treatment solution, 20-�Lroplets of larval suspension were pipetted from the cupnto a microscope slide. All larvae (dead and alive) wereounted using 100× power. Approximately 15–40 lar-ae were then transferred by pipette onto the individualcreens in the MultiScreen filter plate. Larvae that didot get transferred from each droplet on the microscopelide were subtracted from the total number of larvaelaced onto the screen. After at least 15 larvae were placednto the screen, the total well volume was brought up to50 �L with test solution as needed. MultiScreen platesere gassed with CO2 and incubated for 16 h at 37 ◦C. After
he plates incubated, screens were removed and the entireolution of each well of the tear-drop plate was transferredy pipette onto a microscope slide and all larvae wereounted using 100× power.
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.6. Experimental design
Trial 1 was conducted in a completely randomizedesign to determine if Tifton 85 Bermudagrass hay (T85),
PRESSsitology xxx (2013) xxx– xxx 3
dried juniper (DRY), or IVM (40 �g/mL) reduced larvalmotility. Treatments were anaerobically incubated in DaisyII incubator for 18 h in a rumen fluid:McDougall’s buffermixture (1:4). After incubation, 25 mL of treatment solu-tion was centrifuged at 2415 × g for 4 min. Five millilitersof supernatant were added to individual 100-mL plasticcups and ensheathed larvae (n = 1000) were introducedto the treatment solution. To provide a positive con-trol, a treatment cup was incubated with 4.76 mL rumenfluid, ensheathed larvae (n = 1000), IVM (40 �g/mL) and 1%DMSO. All treatment cups were anaerobically incubatedfor 4 h at 37 ◦C. As previously described (Whitney et al.,2011), approximately 35 larvae were then transferred ontoa screen within a multi-screen 96 well plate which con-tained the same treatment and rumen fluid mixture (9wells/treatment). Well plates were gassed with CO2 for30 s, then placed in an airtight bag and gassed for 15 s.Screens were removed 14 h later, and LM was determined;(larvae that migrated through the screen/larvae placedonto screen) × 100.
Trial 2 was used to determine if DRY, OIL, or fresh juniper(FRE) treatment solutions reduced LM. The CNTL (no mate-rial), DRY (7.24%, w/v), OIL (0.334%, v/v; mixed with 4%DMSO), or FRE (11.46%, w/v) were incubated in 800 mL ofbuffer and 200 mL of ruminal fluid for 16 h. Laval motilitywas evaluated after larvae were incubated in this solutionfor 4 h. A positive control (quebracho tannin; 2.5%, w/v)was similarly evaluated and larval motility remained lessthan 5%; quebracho tannin concentration was determinedaccording to Athanasiadou et al. (2001). Twenty-sevenreplicates per treatment were evaluated using 3 runs (3cups/run and 3 wells/cup).
Trial 3 used a 2 × 5 factorial design (CNTL or OIL treat-ment ruminal fluid and 5 IVM concentrations) to determineif larvae initially incubated in OIL would be more suscep-tible to IVM. The CNTL or OIL (0.334%, v/v; mixed with 1%DMSO) treatments were incubated in 400 mL of buffer and100 mL of ruminal fluid for 16 h. Larvae were then incu-bated in this solution for an additional 2 h; thus, CNTL wasincubated for a total of 4 h as in the previous Trials. Mul-tiple IVM concentrations (0, 0.1, 1, 3, and 6 �g/mL; mixedwith 1% DMSO) were then added to these larvae, whichincubated an additional 2 h before LM was evaluated. AnIVM concentration of 45 �g/mL mixed with 1% DMSO (pos-itive control) was similarly evaluated and larval motilityremained less than 4%. Six replicates per treatment wereevaluated using 2 runs (1 cup/run and 3 wells/cup). Trial4 was identical to Trial 3, but used a 3 × 5 factorial design(CNTL, DRY, and FRE treatments and 0, 0.1, 1, 3, and 6 �g/mLIVM) and added DMSO only to IVM. The positive control(45 �g/mL IVM in 1% DMSO) resulted in <0.6% LM. Ninereplicates per treatment were evaluated using 3 runs (1cup/run and 3 wells/cup).
2.7. Statistical analyses
Data were analyzed using the MIXED procedure (SAS
al., Effect of using redberry juniper (Juniperus pin-d increase ivermectin efficacy. Vet. Parasitol. (2013),
Inst. Inc., Cary, NC). All Trials used a model that includedtreatment with run as the repeated measure and wellwithin cup as the subject. For Trial 1, treatment effectswere separated using the PDIFF procedure of SAS. For Trial
Fig. 1. Trial 2: percent larval motility of H. contortus incubated in rumi-nal fluid, buffer, and either no material (CNTL), dried juniper (DRY), freshjuniper (FRE), or distilled juniper terpenoid oil (OIL). The combined aver-age larval motility of DRY, OIL, and FRESH were less (P < 0.001) than CNTL.
Ivermectin Concentration, µg/ml
0.0 0.1 1.0 3.0 6.0
La
rva
e V
iab
ility
, %
20
40
60
80
CONTROL OIL
Fig. 2. Trial 3: percent larval motility of H. contortus incubated in ruminalfluid, buffer, and either no material (CNTL) or distilled juniper terpenoidoil (OIL), and then treated with ivermectin. A treatment × IVM interac-
LM linearly decreased at similar (P = 0.54) rates among
Larval motility of DRY and FRE were similar (P = 0.44).
2, treatment effects were tested using the following singledegree of freedom non-orthogonal contrasts: (1) control vs.average of dried juniper, fresh juniper, and oil; (2) controlvs. average of dried and fresh juniper; and (3) dried junipervs. fresh juniper. Trials 3 and 4 were analyzed using a modelthat included treatment, IVM dose, and treatment × IVMdose interaction. The slice option of SAS was used whentreatment × IVM dose interaction was observed, and lin-ear slopes were compared between treatments accordingto Littell et al. (2006). Data are reported as least squaresmeans with standard errors.
3. Results and discussion
The fresh juniper contained 66.1% DM, 7.18% CP, 43.8%NDF, 42.2% ADF, and 3.5% ash (dm-basis). The dried junipercontained 93.8% DM, 6.6% CP, 39.3% NDF, 38.3% ADF, and3.2% ash (dm-basis). As previously described (Whitneyet al., 2011), percentages of certain individual terpenoids inthe total amount of distilled oil were different from dry andfresh juniper and dried juniper had less total terpenoidsthan fresh juniper. Certain monoterpenoids are volatile andcan be affected by temperature (Animut et al., 2004; Utsumiet al., 2006). Fresh juniper leaf and stem material con-tained 7.3% CT (dm-basis), which consisted of 6.1%, 1.1%,and 0.1% extractable, protein-bound, and fiber-bound tan-nins, respectively. Barry and Manley (1986) suggested thatextractable CT are more reactive than bound CT; thus, theextractable CT concentration in juniper may increase itsanthelmintic properties.
In Trial 1, larval motility for CNTL, T85, DRY, and IVMwere 61.0%, 49.7%, 33.6%, and 26.8%, respectively. Larvalmotility was similar (P = 0.12) when larvae were incubatedin CNTL or T85, suggesting that either CNTL or T85 can beused as a control treatment. Larvae incubated in DRY had
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similar (P = 0.76) motility compared to larvae incubated inIVM, but were less (P < 0.02) motile than larvae incubatedin CNTL or T85.
tion (P = 0.02) was observed due to OIL decreasing IVM efficacy at IVMconcentrations of 1 (P = 0.07), 3 and 6 (P < 0.002) �g/mL.
Adding DRY, FRE, or OIL to ruminal fluid–buffer solutionreduced (P < 0.001; Trial 2; Fig. 1) average larval motility ascompared to larvae incubated in CNTL. The reduction in lar-val motility is probably attributed mostly to the juniper CTconcentration. Min and Hart (2003) found that CT extractedfrom various forages decreased larval motility of severalnematodes including H. contortus. Larvae incubated in OILhad less motility than larvae incubated in CNTL, but resultscould be due to DMSO concentration vs. a true treatmenteffect. In our laboratory, larval motility was approximately80% less when 4% DMSO was used vs. 1% DMSO (Whitney,unpublished data). In addition, larvae incubated in DRY orFRE had similar (P = 0.44) motility, which further supportsthe fact that CT affected motility and not terpenoids. If ter-penoids negatively affected larvae, then larvae incubatedin FRE would be expected to have less motility than larvaeincubated in DRY due to synergistic effects of CT and ter-penoids. Another, less likely, scenario is that terpenoids inFRE did not mix as well as OIL because DMSO was only usedin OIL treatment.
During Trial 3, a treatment × IVM dose interaction(P = 0.02; CNTL and OIL; Fig. 2) was observed due toOIL unexpectedly decreasing IVM efficacy at IVM con-centrations of 1 (P = 0.07), 3, and 6 (P < 0.002) �g/mL.In addition, as IVM dose increased, motility of larvaeincubated in CNTL or OIL linearly decreased at differentrates (P < 0.001). Lei et al. (2010) reported that specificmonoterpenoids were toxic to Caenorhabditis elegans andAscaris suum nematodes. Results of the current study wereprobably not related to OIL being hydrophobic becauseDMSO was used in the OIL treatment and each IVMdose.
No treatment × IVM dose interaction (P = 0.57; CNTL,DRY, and FRE; Fig. 3) was observed during Trial 4, and
al., Effect of using redberry juniper (Juniperus pin-d increase ivermectin efficacy. Vet. Parasitol. (2013),
treatments. However, larvae incubated in DRY ruminalfluid–buffer solution had less (P < 0.004) average motilitythan larvae incubated in CNTL or FRE ruminal fluid–buffer
Fig. 3. Trial 4: percent larval motility of H. contortus incubated in ruminalfluid, buffer, and either no material (CNTL), dried juniper (DRY), or freshj(aa
si
4
mmmrbawftNtrl
A
FDRCpwRB
R
A
A
uniper (FRE), and then treated with IVM. No treatment × IVM interactionP = 0.57) was observed, but larvae incubate in DRY had less (P < 0.004)verage motility than larvae incubated in CNTL or FRE, when averagedcross all IVM concentrations.
olution. Results suggest that incubating larvae in DRYncreased in vitro IVM efficacy.
. Conclusions
For the LMI assay, a control treatment can have either noaterial or Tifton hay included in the ruminal fluid:bufferixture. Dried and fresh juniper material reduced larvalotility, but only dried juniper increased IVM efficacy. The
eduction in IVM efficacy due to larvae initially being incu-ated in OIL was exactly opposite of what was expectednd is currently unexplainable. Further in vivo research isarranted to determine if feeding dried juniper in mixed
eeds to sheep and goats can reduce in vivo H. contor-us motility, fecal egg shedding and increase IVM efficacy.umerous benefits such as re-instating non-effective syn-
hetic anthelmintics by increasing their efficacy would beealized if feeding sheep and goats dry juniper can weakenarvae in the host prior to drenching.
cknowledgements
This study was funded in part by the Food andibers Research Grant Program administered by the Texasepartment of Agriculture. Appreciation is expressed toick Estell (USDA-ARS Jornada Experimental Range, Lasruces, NM) for analyzing samples for percentage of ter-enoids. Some of the L3 Haemonchus contortus larvaeere kindly donated by Dr. Anne Zajac, Virginia-Marylandegional College of Veterinary Medicine at Virginia Tech,lacksburg.
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