Oorsig/Review

Anthelmintic resistance in gastrointestinal nematodes of alpacas (Vicugna pacos) in Australia

Mohammed H. Rashid1, Jane L. Vaughan2, Mark A. Stevenson1, Angus J. D. Campbell1, Ian Beveridge1 and Abdul Jabbar1*

BACKGROUND Australian alpaca industry is an important In the last three decades, the farming of emerging livestock industry. However, very little domesticated South American camelids (SACs), alpacas (Vicugna pacos) and llamas (Lama glama) has increased in Australia, Europe, New Zealand, is known about the epidemiology and control of wormGINs in(Haemonchus alpacas in Australia. contortus) Recently, was reported the first2. 1,2. case of ivermectin resistance in the Barber’s pole Inthe anUK andintensive the USA, farming due to theirsystem, high-quality alpacas fibreand Australian alpaca farmers revealed that the dose and adaptability to many climatic conditions ofA surveyanthelmintics of the worm used controlfor alpacas practices (e.g. usedone byto common in domestic ruminants; e.g. Haemonchus three times of dose recommended for sheep) and contortusllamas can, Ostertagia be infected ostertagi with both, Trichostrongylus shared (those the existence of other potential risk factors for spp. and Nematodirus spp.) as well as host- the development of AR known for GINs of sheep, Lamanema chavezi) gastrointestinal goats and cattle, could lead to the development of nematodes (GINs)3–5 AR in GINs of alpacas (Rashid et al specific (e.g. data). economic losses from that lowered can cause production significant of , unpublished clinical and subclinical problems,3, 4, 6–8. resulting in The aims of this study were (i) to undertake

fibre,Outside meat South and/or America, leather knowledge on the parasites of SACs is limited. Traditionally, the use witha questionnaire the development survey of to AR obtain in GINs insights of alpacas into farmlevel characteristics that might be associated commonly used method to treat and control anthelmintics against GINs of alpacas in Australia. GINsof chemotherapeutic of domestic ruminants. agents has Similarly, been the farmers most and (ii) to quantify the efficacy of commonly used regularly use various classes of anthelmintics to METHODS control GINs in alpacas and llamas3, 4, although no anthelmintic is registered for use against GINs in SACs in Australia. Given that very little is known Study population 9, farming in Australia mainly occurs in four Australia has various climatic zones, and alpaca registeredabout the pharmacokineticsfor domestic ruminants of drugs at indifferent SACs dosethe off-labelrates recommended use of anthelmintics for goats, insheep alpacas and alpacazones, theherds Mediterranean, are located non-seasonalin the south-eastern rainfall, cattle is commonplace. However, the dose rate(s) statessummer of rainfallNew South and winter Wales rainfalland Victoria, zones. Mostwith and route(s) of administration recommended for fewer in Queensland, Western Australia, South Australia and Tasmania. The majority of alpaca SACs10 as found previously in goats11. Thus, under-dosingsheep might of not anthelmintics be effective may against promote GINs the in development of anthelmintic resistance (AR) in supplementaryfarms contain ≤feed 50 animals,(Rashid et which al graze year- data).round Alpacas on pastures, are routinely with variable vaccinated provision against of of the risk factors for the development of AR in ; unpublishedClostridium GINs ofin sheepSACs as12. Caseunder-dosing reports of is ARin known GINs to of be SACs one perfringens type D, C. tetani, C. novyi type B, 2, Belgium13, C.clostridial septicum and diseases C. chauvoei (caused). They by are generally Canada14 and the USA15 in various GINs against shorn once annually in spring, although at twohave commonly been reported used from classes Australia of anthelmintics,

Australia has the largest alpaca population variable timesthroughout the year. Timing and benzimidazoles and macrocyclic 16 lactones. (>450,000) outside South America and the duration of the birthing periods vary between farms but often occur during about two months

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Faecal egg count reduction test (FECRT) The FECRT was performed on each farm according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines

ruminants18, 19. Both female and male - Huacaya andfor Suri the alpacas, evaluation aged of 3-months anthelmintic to 16-years efficacy were in randomly selected on each farm and allocated to

and an untreated control group) comprising 5–15 animals.five or six Six groups anthelmintics (anthelmintic were treatment evaluated groups in this study: (i) monepantel (Zolvix®, Elanco Pty. Ltd., West Ryde, New South Wales, Australia); (ii) a

Fig. 1 Map of Australia showing the locations of alpaca farms enrolled in the faecal egg count reduction trials in this study. Each combination of levamisole, closantel, albendazole circle represents one alpaca farm Rutherford, New South Wales, Australia); (iii) closanteland abamectin with sodium (Q-drench®, selenate Jurox (Closicare Pty. Ltd.,Plus in spring. Crias are weaned at an average age of Wales, Australia); (iv) ivermectin (Ivomec®, three months. BoehringerSelenium®, VirbacIngelheim Pty. Ltd.,Pty. Milperra,Ltd., North New SouthRyde, New South Wales, Australia); (v) moxidectin Questionnaire survey The survey aimed to assess current worm control practices of Australian alpaca farmers, (Cydectin® Injection for cattle, Virbac, Pty. Ltd., with an emphasis on the use of anthelmintics. BendigoMilperra, East, New Victoria, South Wales, Australia). Australia); Resistance and (vi) to A questionnaire was conducted using an online ivermectinfenbendazole was (Panacur found up 25®, to the Intervet 9th FECR Pty. trial. Ltd., programme, Research Electronic Data Capture17. Hence, we decided to replace ivermectin with a

FECR trials. All anthelmintics were administered practices;The questionnaire (ii) the usecontained of anthelmintics; 30 questions and about (iii) more potent macrocyclic lactone in subsequent (i) farm demography and general husbandry 1.5 times the dose rate recommended for sheep. were close-ended, with a few semi-open (i.e. orally apart from moxidectin (subcutaneously) at agrazing close-ended management. question The with majority the ofaddition questions of a category “other”). An online questionnaire faecalAnimals samples were dosed were individuallycollected from based the on rectum body survey was supplied to 97 alpaca farmers who pre-weight (day using 0) and scales post-treatment where available. (day 11–14) Individual into had responded to a larger survey on more general parasite management in Australia (Rashid et al; methodzip-lock plastic[20] within bags, andseven were days kept of at collection. 4 °C until aspects of alpaca husbandry, worm problems and processed for FEC using a modified McMaster of water added into a 60ml container and soaked Selectionunpublished of farmsdata). Briefly, four grams of faeces were mixed with 11 ml Out of 91 alpaca farms that responded to the survey, 20 farms were selected to take part in 1.27)for 5–30 solution min (45 before ml) was making added a and homogenised 30–45 min later,faecal the slurry. sample Saturated was agitated, sugar (specific and a sample gravity drawn immediately from the suspension using a theirfaecal herd egg count(Fig. 1). reduction The following testing selection (FECRT) criteria based on herd size and the geographical location of 40 and 60 alpacas of different ages and sexes; sieve-top pipette (sieve aperture size 12 meshes were used: (i) the herd was comprised of between per cm), to fill two chambers of a Whitlock egg the 8 weeks prior to the scheduled herd visit; thecounting slide was slide placed (http://www.whitlock.com.au/ on the stage of a compound (ii) deworming had not been carried out within lightslides/JAWCO_Home.htm). microscope and eggs Afterwere fivecounted. minutes, The (FEC) were greater than or equal to 150 eggs per gram(iii) confirmation (epg) of faeces; that and average (iv) there faecal was egg a historycounts EPG. sensitivity of the McMaster technique was 15 years. When a farmer agreed to participate and Nematode identification of anthelmintic usage on the farm in the last five diagnostic kit (Easy-Plex, AusDiagnostics andmet tested the first for twoFEC. criteria,Over 50 faecalfarms were samples tested were to We used a newly established molecular collected from fifteen randomly selected alpacas 21. Pty. Ltd., Beaconsfield, Australia) for the obtain 20 suitable farms for the FECRT trial. identification of common GINs of alpacas

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Faecal DNA was extracted using a method 22. Preand post-treatment faecal samples for each genomicFor primary DNA amplification representing (15each cycles DNA ofsample 10 s or at described previously with few modifications 95 °C, 20 s at 60 °C, and 20 s at 72 °C), 5 μl of sample representing 5–15 individual faecal into 0.2 ml PCR strips and placed into a 24-well samplesgroup were per pooledgroup. inThis order was to achieved obtain one during DNA 5 μl of water (negative control) were dispensed processing of individual faecal samples for FEC thermocycling block in the High-Plex 24 system in the saturated sugar solution to a 50 ml Falcon (AusDiagnostics). Subsequently, the analysis by withdrawing 1 ml of the faeces suspension was executed by the program MT-Assay Setup per treatment group. theSoftware melting (AusDiagnostics). curve analysis were Following performed the first in tube. This process was repeated for each sample round of PCR, the secondary amplification and Software (AusDiagnostics). a 96-well MT-Analyzer using the MT Analysis FollowingFinally, the centrifugation Falcon tube (2500× was filled rpm, by 10 adding min), Each sample was recorded as test-positive using themore supernatant floatation (~5 solution ml) was to makecollected 50 and ml. the auto-call function of the Easy-Plex software (AusDiagnostics) if the amplicon produced a

taptransferred water was to added another to 5050 mlml Falconand centrifuged tube. To the expected melting temperature, the height (2500×wash the rpm, GIN eggs10 min).collected The in supernatant the Falcon tube,was single melting curve which was within 1.5 °C of discarded and the pellet was washed twice more, of the peak was higher than 0.2 dF/dT (where pooled eggs with remaining faecal materials otherwisedF/dT is the the sample derivative was considered of fluorescence as negative over using the same steps as above. The washed (AusDiagnostics).temperature), and Additionally,the peak width cycle was threshold ≤ 3.5 °C, (Ct) values for each nematode per sample were transferred into a microcentrifuge tube faecaland stored material at -20 was °C useduntil furtherto extract use. and Following isolate thawing, a 250 μl sample of the concentrated were determined by comparing with the data obtained from the internal spike control which DNA using Powersoil® DNA purification kit had a known 10,000 DNA copy numbers. Based The(MoBio, assay USA) was as conductedper manufacturer’s in the High-Plex protocol. 24 on the peak high-resolution melting (HRM) temperatures.temperature analyses, Randomly nematode selected genera/species amplicons were assigned according to their mean HRM system with the MT-Assay Setup Software for the first round of PCR and the 96-well MT- nematodes.representing each nematode genus/species Wales,Analyzer Australia) and the for MT the Analysis second Softwareround of (Cat.PCR. were subjected to sequencing to verify the target No. 9150, AusDiagnostics, Mascot, New South Statistical analyses Questionnaire data were downloaded from primerThe primary pairs amplificationdesigned for the(‘target sequences enrichment’) of the was conducted using nematode-specific REDCap as a comma-separated values (CSV) file. AusDiagnostics].Internal Transcribed Spacer 2 (ITS 2) [Step 1 eggData count validation reduction and (FECR)cleaning was were performed performed using by tubes for nematodes (8-well), Cat. No. 78150S, using Microsoft Excel 2013. Calculation of23 faecal and following the WAAVP guidelines19, 24. FECR (%) the contributed R package “eggsCount” pairsThe to the secondary internal regions amplification of the ITS 2 (Alpaca for group at post-treatment collection for each semiquantification employed nested primer anthelmintic.was calculated Faecal between egg count treatment data are and generally control Camelostrongylus over-dispersed and inherit Poisson errors, which mentulatusNematodes, MP96Cooperia 8-well, spp., Cat.Haemonchus No. 78150E, spp., was incorporated in the R package “eggsCount”24. OstertagiaAusDiagnostics) ostertagi specific, Oesophagostomum to spp., Thus, it has advantages over Excel spreadsheets Teladorsagia circumcincta and Trichostrongylus spp. These internal primer pairs amplify a inherent errors of faecal egg count data into account.to calculate FECR more precisely by taking the Furthermore, another primer pair was included region of ~90 to 110 bp from the ITS 2 region. Interpretation of the FECRT results Anthelmintic resistance status was interpreted in each run as a reference to assess the efficiency of amplification from 10,000 copies of a synthetic oligonucleotide template (internal ‘spike reductionas recommended (%FECR) by and the the WAAVP upper guidelines (UCL) and on control’). AR based on the percentage of faecal egg count

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18. Hence, each anthelmintic was declared as (i) effective when lower (LCL) 95% confidence limits tested the efficacy of anthelmintics in use on their Faecalfarms using egg count FECRT reduction (Table 1). tests %FECRthe %FECR was and< 95% UCL or were LCL bothwas < ≥ 90%, 95% and and (iii) the LCL was ≥ 90%, (ii) suspected resistant when 95% and LCL was < 90%. Furthermore, multiple The FECRT results revealed that a combination of ARineffective/resistant was declared when when parasite both %FECRpopulations was of < levamisole, closantel, albendazole and abamectin was the most effective dewormer (78%, 14/18 susceptible farms), followed by monepantel classesGINs were25. identified, using the above criteria, to (75%, 15/20), moxidectin (27%, 3/11), closantel be resistant to anthelmintics of different chemical (5%, 1/20), fenbendazole (0%, 0/19) and RESULTS ivermectin (0%, 0/10) (Figs. 2 and 3; Table 2; see alpacasAdditional showed file 1:the Table presence S1). Theof mixed molecular GIN identification of GINs from all herds of Questionnaire survey infections containing C. mentulatus, Cooperia spp., The response rate for the questionnaire was 94% Haemonchus spp., O. ostertagi, Oesophagostomum spp., T. circumcincta and Trichostrongylus spp. Haemonchus spp. (49%) were the most (91/ 97). Huacaya was the more popular alpaca Table 1 Worm control practices used by Australian alpaca farms breed, with an average herd size of 77 (minimum included in this study other9; maximum livestock 600) species alpacas. such as About sheep half and ofcattle, the Responses respondents (51%, 46/91) kept alpacas with Worm control factor No. of responses (%) and 26% (24/91) of alpaca farmers allowed their Keeping alpacas with other animals to co-graze with other domestic livestock species 51 ruminants (Table 1). Twenty five percent (23/91) alpacas on their farms at least once during the last 46/91 of respondents had agisted non-home-bred livestock species 26 Co-grazing of alpacas with other reported that worms were an important Keeping agisted alpacas 24/91 25 healthfive years. issue Although for their 63% alpacas, (57/ 91) the of respondentsmajority of Worms is an important health 23/91 issue of alpacas 63

Anthelmintic usage 57/91 89 respondents (89%, 81/91) used anthelmintics Types of anthelmintics used Thefor the commonly control of GINsused in theiranthelmintics animals (Table were 1). 81/91 macrocyclic lactones (e.g. ivermectin, moxidectin; 65 Macrocyclic lactones (MLs) 53/81 31 Monepantel 25/81 20 65% (53/81)), monepantel (31%, 25/81), Levamisole (LEV) 15 benzimidazoles (BZs) (20%, 16/81), levamisole Benzimidazole (BZ) 16/81 Closantel 10 (LEV) (15%, 12/81), closantel (10%, 8/81), 12/81 and their combinations, including two [BZ and 8/81 (BZ and LEV) 7 MLs, 7% (6/81)], three [BZ, LEV and MLs, 9% Double combinations (7/81)] or four [closantel, BZ, LEV and MLs, 37% 6/81 9 anthelmintics(29/81)] anthelmintics at the dose (Table rate recommended 1). The majority for Triple combinations of respondents (53%, 43/81) used these (BZ, LEV and ML) 7/81 sheep, though some used 1.5 times the dose rate of levamisole, closantel, Q-Drench (a combination 37 Deworming time recommended for sheep (23%, 19/81) or the albendazole, abamectin) 29/81 dose rate recommended for cattle (10%, 8/81). Before winter 43 At shearing 35/81 36 The use of anthelmintics seemed to be need-based At weaning 29/81 32 (62%, 50/81) rather than a part of any strategic Dose rate 26/81 deworming program twice (25%, 20/81) or once per year (12%, 10/81). Alpacas were dewormed Sheep dose 53 either before winter (43%, 35/81), at shearing 1.5 times sheep dose 43/81 23 were(36%, using 29/81) one or classat weaning of anthelmintics (32%, 26/81) for (Table more Cattle dose 19/81 10 1). The majority of respondents (60%, 49/81) Testing of anthelmintics 8/81 a 12 than a year, and most of them (72%, 58/81) said forRotation efficacy of (e.g.anthelmintics FECRT) 11/91 72 that they changed (‘rotated’) drench classes in the last five years. A relatively small proportion a FECRT faecal egg count reduction test58/81 of respondents (12%, 11/91) reported that they

7 Oorsig/Review

Trichostrongylus spp. (37%), C. mentulatus preand post-treatment pooled faecal samples (28%),commonly O. ostertagi resistant (25%) nematodes and Cooperia followed spp. by ofMolecular alpacas detectionrevealed ofthat seven monepantel common GINsand ina (13%) whereas Oesophagostomum spp. (100%) and T. circumcincta successful in eliminating all the GINs (i.e. anthelmintics tested in this study (Figs. 4 and 5; C.combination mentulatus of, Cooperia anthelmintics spp., wereHaemonchus most (99%) were susceptible to spp., O. ostertagi, Oesophagostomum spp., T. circumcincta and Trichostrongylus spp.) Ivermectinsee Additional and file moxidectin, 1: Table S2). the two compounds prevalent on 20 alpaca farms (Fig. 4; see Additional

infrequently effective at the dose rates used in Haemonchus spp. and Trichostrongylus spp., with tested as single ML anthelmintics, were veryfile 1: little Table effect S1). on Fenbendazole C. mentulatus had, O. ostertagi no effect and on ivermectin was tested, the FECR on six farms was Cooperia spp. Likewise, ivermectin showed no 2–87%this study and (Fig. there 3; Table was 2).no On reduction the 10 farms in FEC where on Haemonchus spp. the remaining four farms (i.e. FECR -329 to -7%) efficacy against withC. mentulatus variable andeffect O. onostertagi other GINs while (Fig. it was 4). moderately Moxidectin effective seemed the(Fig. remaining3; Table 2). eightConversely, farms, moxidectin with three was farms fully to have the highest efficacy against effective on three farms but was not effective on Closantelagainst other is fivea narrow-spectrum GINs (Fig. 4). anthelmintic anthelmintichaving suspected in this resistance study was and ineffective the other against five GINsresistant of alpacas GINs (Fig. on 3;all Table farms, 2). with BZ used FECR as ranginga single nematodes such as Haemonchus spp. However, it wasand notknown effective to be against effective this against important blood nematode feeding epg (FECR -522 to -18%) on six farms. Similarly, in all alpaca farms studied herein (Fig. 4). between 8–94% on 13 farms, with an increase in only one farm, while resistance and suspected Discussion resistanceGINs were foundto this to drug be susceptible occurred on to 15closantel (FECR onof Gastrointestinal nematodes are a major clinical 29–93%) and four (FECR -194 to -13%) alpaca and economic threat to SACs throughout farms, respectively. the world. Since there are no anthelmintics registered for use in SACs, limited information is

onMonepantel alpaca farms and ina combinationthis study, with of anthelmintics the majority available on their efficacy and safety. This is the were found to be the most effective drenches first study to comprehensively investigate the efficacy of commonly used anthelmintics against twoof prevalent or more anthelmintics GINs ~100% from susceptible different to classes both wasGINs theof alpacas most commonlyand llamas. effectiveThe combination dewormer of areanthelmintics ineffective) (Fig. was 3; detected Table 2). on Multiple all alpaca AR (when farms onlevamisole, the 20 Australian closantel, albendazolealpaca farms and in abamectinthis study,

included in the study (Table 2). followed by monepantel, moxidectin, closantel,

Fig. 2 Overall efficacy of six anthelmintics against gastrointestinal nematodes of alpacas on 20 farms in Australia. Each circle shows percentage of the faecal egg count reduction while each horizontal line shows upper and lower 95% confidence intervals. Abbreviations: CLO, closantel; CYD, cydectin; IVM, ivermectin; FBZ, fenbendazole; QDR, Q-drench (a combination of abamectin, albendazole, closantel and levamisole); MON, monepante

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Fig. 3 The proportion of farms with resistance, suspected resistance and susceptibility of gastrointestinal nematodes of alpacas to six anthelmintics on 20 farms in Australia. Abbreviations: CLO, closantel; CYD, cydectin; IVM, ivermectin; FBZ, fenbendazole; QDR, Q-drench (a combination of abamectin, albendazole, closantel and levamisole); MON, monepantel

were the most commonly resistant nematodes not followed. The FECRT results of this study fenbendazoleTrichostrongylus and ivermectin. spp. Haemonchus, C. mentulatus spp., quarantine deworming and/or procedure(s) are O. ostertagi and Cooperia spp. Previously, AR in various anthelmintics in alpacas at 1.5 times followed by should be interpreted carefully as we tested from Australia2, Belgium13, Canada14 and the USAGINs15 ofagainst alpacas two and commonly llamas had used been classes reported of dosesthe dose of commonly rate recommended used anthelmintics for sheep in because SACs. no information is available on the therapeutic lactones in H. contortus. However, almost all of of treatment for the FECRT on some farms was theseanthelmintics, studies were benzimidazoles case reports, and including macrocyclic the Furthermore, the number of animals per group one from Australia2 as opposed to this study less (see Table 2) than that (10–15 animals in GINs of alpacas at a national level across the GINsper group)of ruminants recommended18 due to lack by theof the WAAVP required for alpacathat provides industry insights in Australia into - the the problemcountry which of AR evaluating the efficacy of anthelmintics against

America. number of animals per farm. Additionally, two has the highest number of alpacas outside South MLs, ivermectin and moxidectin were not tested In this study, the questionnaire survey revealed on all farms as we wanted to assess the efficacy Furthermore,of both a less we potent could (ivermectin) not test these as two well drugs as a practices on Australian alpaca farms. Over 50% onmore any potentfarm together (moxidectin) due to the ML lack drug of inalpacas alpacas. for ofinvaluable farmers kept information alpacas aboutwith sheep the husbandryand cattle the FECRT at any farm. ruminants that can expose alpacas to shared GINsas well such as allowingas Haemonchus them to spp. graze3, 5, 26–28 with, sometimes domestic 30. resulting in fatal infections2, 6. Similarly, keeping AnthelminticFenbendazole resistance and albendazole to BZs are was widely reported used inbroad-spectrum GINs of sheep dewormers from Australia in sheep soon and after cattle the similar GINs on the same property, can increase 31. Recently, Playford thedifferent transmission livestock of species resistant that GINs can harbouramong et al.32 conducted a national survey to assess the ruminants. Previously, Edwards et al.29 found prevalenceintroduction of of AR thiabendazole in GINs of sheep in Australia the highest prevalence of levamisole resistant and they found that 96% of farms in Australia Trichostrongylus spp. in sheep where sheep and had resistant H. contortus, T. circumcincta and Trichostrongylus spp. Similarly, we found that in Western Australia; however, such studies have none of the 20 alpaca farms included in this study cattle had grazed together on the same paddock we found that agistment of alpacas occurred onnot 25% been of undertaken Australian foralpaca SACs. farms Furthermore, surveyed. weight,had susceptible with an overallworms FECR(as per of guidelines36% (see Fig.of the 2, This practice may further increase the risk of WAAVP) against fenbendazole at 7.5 mg/kg body introducing resistant GINs to a herd if proper Table 2).

9 Oorsig/Review

Table 2 Faecal egg count reduction percentages (%FECR) and 95% confidence intervals (CI) calculated by the faecal egg count reduction test (FECRT) 10–14 days after anthelmintic treatments in alpacas naturally infected with gastrointestinal nematodes on 20 alpaca farms in Australia

Farm no. %FECR (95% CI) of anthelmintics Q-drencha Closantel Ivermectin

01 Monepantel100 98 (93–100) Fenbendazole64 (-55–95) 42 (-270–91) MoxidectinNT

02 98 (94–100) NTbNT 54 (-3–80) -60 (-286–33) -329 (-1157–46) NT 03 99 (90–100) 100 (94–100) 86 (17–98) NT 40 (-224–87) NT 04 99 (87–100) 99 (96–100) 93 (66–98) 87 (34–97) 45 (-217–91) NT 05 100 100 92 (72–97) 56 (-44–87) -58 (-589–63) NT 06 95 (75–99) 100 25 (-168–79) 44 (-250–91) -31 (-293–56) NT 07 98 (78–100) 100 80 (-1–96) 8 (-254–76) 21 (-269–83) NT 08 100 100 34 (-32–67) 58 (11–80) 87 (54–97) NT 09 100 100 -65 (-1291–80) -522 (-4368–14) -7 (-618–84) NT 10 97 (75–100) 99 (92–100) 29 (-44–65) 55 (-7–81) 2 (-280–74) 39 (-355–92) 11 100 96 (67–100) 66 (10–87) 15 (-201–76) NT 93 (65–98) 12 100 100 97 (81–99) 75 (-59–99) NT 98 (84–100) 13 100 99 (92–100) 90 (47–98) 94 (82–98) NT 99 (85–100) 14 100 99 (86–100) 95 (69–99) -203 (-1396–39) NT 98 (84–100) 15 100 100 49 (-104–87) 70 (13–90) NT 100 16 100 93 (42–99) 96 (79–99) 50 (-174–91) NT 1 (-566–85) 17 100 100 -194 (-1262–37) 9 (-118–62) NT 89 (-2–99) 18 100 100 96 (48–100) 78 (-33–96) NT 90 (23–99) 19 99 (85–100) 99 (85–100) -13 (-327–70) -147 (-848–36) NT 100 20 100 100 -96 (-3257–89) -18 (-1560–92) NT 100

a NT not tested b Q-drench contains levamisole, closantel, albendazole, abamectin spp., O. ostertagi, Oesophagostomum spp. and Haemonchus spp. and Trichostrongylus spp., Trichostrongylus spp. (see Fig. 4). Similar results andIt completely was only failedpartially to reducesuccessful the in number reducing of were found in alpacas and llamas from the USA C. mentulatus, Cooperia spp., et al.15 when they used the same dose O. ostertagi and Oesophagostomum spp. (see rate and the route of administration of ivermectin Fig.the numbers4). Previously, of BZ resistance in GINs of againstby Gillespie H. contortus

(FECR 22–37%) than. However, on one alpaca they found farm (FECRbetter alpacas has been reported from the USA [14] efficacy of ivermectin in llamas on two farms where no FECR (-111%) was observed when et al.2 also found H. contortus in alpacas resistant usedfenbendazole at the same was dose given rate. orally The second at 10 BZ mg/ used kg to-65%). ivermectin In a previous (FECR study35%) from when Australia, given orally Jabbar at compared with 59% when albendazole was with other anthlemintics, levamisole, closantel 45. in this study was albendazole in combination The200 questionnaire μg/kg. Ivermectin survey resistance results inof strongylesthis study most effective anthelmintics against GINs of of alpacas has also been reported from Peru and abamectin, which proved to be one of the therevealed high frequencythat MLs were of the the use most of dewormers commonly usedhave alpacas. The efficacy of BZ in this combination anthelmintics by Australian alpaca farmers, and presenceformulation of effective cannot be anthelmintic(s) evaluated separately when two as of AR in sheep GINs34 orthe more high classesefficacy of of anthelmintics this group might are beused due33 .to the thebeen case found for to the be associated development with of the AR development in GINs of ; hence, this might also be In this study, ivermectin failed to reduce FECs study was 81% effective against GINs of alpacas (-20%, see Fig. 2) of nematodes when given orally atSACs. farm The level, second and ML,resistance moxidectin, was reported used in thison

Haemonchus sp partiallyat 300 μg/kg. effective In addition, against C. it mentulatus was unsuccessful, Cooperia in of46% moxidectin farms, with resistance 27% farms against susceptible GINs in and alpacas. 27% reducing the numbers of p. but was suspected for resistance. This is the first report

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However, Gillespie et al.15 reported moxidectin spectrum anthelmintic and is recommended resistance against H. contortus in llamas from the for H. contortus in small ruminants in Australia. USA. Findings of this study showed that 75% of the 20 alpaca farms had resistant populations of

The third ML used in this study was abamectin againstGINs. However, the majority this result of shouldGINs ( C.be mentulatusinterpreted, in combination with other anthlemintics, Cooperiacarefully asspp closantel., O. ostertagi is not claimed, Oesophagostomum to be effective againstalbendazole, GINs of levamisole alpacas. Closantel and closantel is a narrow which spp., T. circumcincta and Trichostrongylus spp.) proved to be one of the effective anthelmintics

Fig. 4 Gastrointestinal nematodes found in faeces of alpacas pre- and post-treatment with six anthelmintics on alpaca farms in Australia. The nematodes were identified using the multiplexed-tandem PCR. Abbreviations: CLO, closantel; CON, negative control; CYD, cydectin; IVM, ivermectin; FBZ, fenbendazole; QDR, Q-drench (a combination of abamectin, albendazole, closantel and levamisole); MON, monepante

11 Oorsig/Review

found in alpacas on the 20 farms. Regarding its t Haemonchus spp., this nematode on different Australian alpaca farms (see Fig o fenbendazole, closantel and ivermectin efficacy against when two or more classes of anthelmintics are was found on 18 (out of 20) farmset but al .32closantel where 4). Multiple anthelmintic resistance occurs was effective only on 50% (9/18) farms. Similar closantelresults were resistance found in by H. Playfordcontortus of sheep from tounable anthelmintics to control at GIN their populations therapeutic that doses were they reported 43% (23/53)et al .2 prevalence found that an of [40].previously Previously, susceptible Gillespie (more et thanal.15 documented 95% killed) ivermectin-resistant population of H. contortus in H. contortus resistance to ivermectin, Australia. Conversely, Jabbar

Givenalpacas thatwas 99%H. contortussusceptible can to closantel. lead to fatal commonfenbendazole among and the moxidectin main GINs inthat llamas infect from sheep the40, infections in alpacas and it is resistant to most goatsUSA. Multiple41 and cattle resistance42. Given to that anthelmintics Australia is amongis very of the commonly used anthelmintics, closantel the world leaders for the high prevalence of multiple AR in GINs of small ruminants32, more

should be used very carefully in areas/farms where its resistance has not been reported, AR.sustainable strategies to control GINs in alpacas thereby delaying the development of AR against are required to control the problem of multiple monepantelspecific, narrow against spectrum GINs in alpacas. anthelmintics. We did This not is the first report documenting the efficacy of This study utilised a newly established molecular asfind per monepantel the guidelines resistance of the WAAVP, in GINs monepantel of alpacas post-treatmentdiagnostic technique pooled (MT-PCR)faecal DNA to samples identify21. when used at 3.75 mg/kg body weight. However, Traditionally,nematode genus/ larval species cultures of alpacas (LC) are in pre-used and to 2). resistance was suspected on five farms (see Fig. this procedure is time-consuming and lacks Previously, Dadak et al.10 undertook a study identify nematode genus/species. However, requires experienced personnel for accurate monepantel for treating GINs in llamas in Austria, sensitivity and specificity. In addition, the LC aimed at establishing an effective dose rate of morphologicallyidentification of 43 the. third-stage larvae (L3s) as and found that three dose rates of 2.5 mg/kg, many nematode species are difficult to distinguish respectively.5.0 mg/kg and However, 7.5 mg/kg we used of monepantel1.5 times the weredose rateable recommended to reduce FECs for by sheep 84%, herein 93% as and it was 100%, the nematodesThe MT-PCR ofassay alpacas, used in C. this mentulatus study also21 allowed as LC an accurate identification of one of the common most commonly used dose by Australian alpaca farmers as well as veterinarians (J. Vaughan, that monepantel at this dose rate was 100% stagedoes notlarvae. allow Therefore, its reliable the identification testing of pre- due and to unpublished data). In addition, we found effective against GINs on 13 alpaca farms while post-treatmentunavailability of pooled morphological faecal DNA keys samples of the thirdfrom

95–99% effective on seven farms (see Table 2). in two species of SACs using different doses ivermectin,20 alpaca farms monepantel allowed us, moxidectin for the first time,and toa These differences in the efficacy of monepantel ascertain the efficacy of closantel, fenbendazole, pharmacokinetic properties of the drug in alpacas C. mentulatus as well as other GINs (see Fig. 4). andmight llamas. be associated with differences in the combination of four anthelmintics against epidemiological and AR studies to accurately However, this proposal warrants further identifyHence, the the MT-PCR common assay GINs should of alpacas be used as in well future as investigation. Although monepantel is a relatively llamas. new drug, GINs of sheep resistant to this drug 35 36, New Zealand37, Netherlands38 and Uruguay39. Similarly,have been the reported injudicious from and Australia frequent, Braziluse of We assessed the efficacy of a combination of monepantel can also lead to the development dewormerfour classes inof anthelminticsalpacas in this for study.the first Recently, time in of AR in GINs of alpacas. Therefore, care should alpacas and it was found to be the most effective

controlthere has of beenGINs of growing ruminants interest as multiple in the useAR ofis be taken when using monepantel to prolong its ancombinations emerging threat of anthelmintic for the control classes of nematode for the efficacy against GINs of alpacas as this is one of parasites33, 44, 45. Given that we found multiple the two dewormers found to be effective herein. This study presents multiple AR in GINs of alpacas AR for all anthelmintics when used as a single

for the first time as most of them were resistant 12 Volume 23 • Issue 05 • 2019

Fig. 5 Percentage of resistant gastrointestinal nematodes against commonly used anthelmintics on 20 alpaca farms in Australia

that the serum concentration of ivermectin in alpacas after the administration via various dewormer, the use of combinations of two or delayingmore anthelmintics the development with goodof AR inefficacy GINs of as alpacas single anddewormer(s) llamas. However, could be future an effectivelarge-scale means studies of onroutes pharmacokinetics will be very similarof anthelmintics to those foundin SACs in complicatellamas. Such the situation limited and but do conflicting not provide reports sound of anthelmintics against GINs of SACs. will be required to test a variety of combinations dose rate and route(s) of administration for Given that no anthelmintics are registered for use differentevidence-based anthelmintics practice in for alpaca using anand accurate llama medicine. Therefore, large-scale pharmacokinetic pharmacokinetic properties of the commonly studies are needed to understand usedagainst anthelmintics GINs in SACs andin veryalpacas little and is known llamas, about an pharmacokinetic properties, appropriate dose appropriate dose rates and the route(s) of rate(s) and the route(s) of administration of the administration for various anthelmintics in these commonly used anthelmintics in alpacas and animals are unknown. For example, Guerden & llamas. Hemelrijk46 found that ivermectin reduced 100% CONCLUSIONS FECs of GINs in both alpacas andet llamasal.8 reported when used subcutaneously at a dose rate of 200 μg/kg body weight. However, Windsor This is the first study to comprehensively infectionsthat subcutaneous in alpacas. administration of ivermectin anthelmintics against GINs of alpacas and reduced but did not completely eliminate GIN investigate the efficacy of commonly used Conversely, the oral administration of ivermectin mostllamas. commonly The combinationeffective dewormer of levamisole,on the 20 closantel, albendazole and abamectin was the result in the reduction of GINs of alpacas in the USAat a dose(FECR rate -65%) of 30015 or μg/kg in Australia body weight (FECR did -20%, not andAustralian ivermectin. alpaca Haemonchus farms in this spp study,. were followed the most by monepantel, moxidectin, closantel, fenbendazole duethis to study). the different These differencesroute of administration in the efficacy (oral of Trichostrongylus spp., C. mentulatus, O. ostertagi ivermectin in the above four studies might be andcommonly Cooperia resistant spp. This nematodesstudy highlights followed the need by of ivermectin was lower in llamas following its for future large-scale pharmacokinetic studies to vs subcutaneous) as the serum concentration understand pharmacokinetic properties, appropriate dose rate(s) and the route(s) administration at a dose rate17. However, of 200 μg/kg Burkholder body of administration of the commonly used anthelmintics in SACs. etweight al.47 orally (less than 2 ng/ml) than ivermectinsubcutaneously in serum (3 ng/ml) of llamas when ivermectin were not able to find detectable levels of References available online: www.vetlink.co.za was injected subcutaneously at a dose rate of 200 Theμg/kg pharmacokinetics body weight. of ivermectin have not been studied in alpacas but we cautiously expect

13 Oorsig/Review CPD AC/2275/19 SMS code: a93613

01. Cria’s in this study were weaned at what 07. Closantel is a narrow spectrum anthelmintic average age? and is recommended for use against which species of intestinal worm in small ruminants? a. 6 weeks a. H. contortus c. 5 months C. metulatus d.b. 36 months c. O. ostertagi e. 8 months d.b. Cooperia spp e. Trychostrongylus spp 02. Which anthelmintic was withdrawn from the trial since resistance was found up to the 9th 08. Which single anthelmintic administered Faecal Egg Count Reduction Tests (FECRT)? orally at 1.5x the recommended sheep dosage in this study was 100% effective against gastro- a. Closantel intestinal worms on 13 farms and 95-99% effective on the other 7 farms? c. Ivermectin b. Cydectin a. Levamisole

d. Fenbendazole 03.e. Monepantel Anthelmintics were administered orally at b. Ivermectin what dosage during the trial? c. Monepantel d. Fenbendazole a. Dosage recommended for sheep 09.e. Abamectin Which of the following statements is false?

c. 3x the sheep dosage a. Traditionally, larval cultures are used to identify d.b. Dosage2x the cattle recommended dosage for cattle e. 1.5x the sheep dosage is time-consuming and lacks sensitivity and nematode genus/species. However, this procedure 04. Which worm species was found to the most commonly resistant to anthelmintics? specificity b. (L3s)Larval cultures require experienced personnel for a. Haemonchus accurate identification of the third-stage larvae Trichostrongylus c. Ostertagia c. nematodeThe MT-PCR of assayalpacas, used C. mentulatusin this study also d.b. Cooperia allowed an accurate identification of the common e. Oesophagostomum C. mentulatus due to d. Larval cultures do not allow the reliable 05. The most effective dewormer in this study stageidentification larvae of was found to be? unavailability of morphological keys of the third- faecal samples a. Levamisole e. PCR cannot be used to identify nematodes in 10. Which one of the following statements is false

b. Closantel c. Albendazole pharmacokinetics of anthelmintics in South d. Abamectin a. LimitedAmerican but camelids conflicting complicate reports onthe situation and 06.e. A Which combination country of hasall of the the highest above number of Alpacas outside South America? for using an accurate dose rate and route(s) of administrationdo not provide soundfor different evidence-based anthelmintics practice in a. China alpaca and llama medicine

c. USA American Camelids d.b. MongoliaAustralia b. Llamas and Alpacas are commonly known as South e. France c. productionA baby Alpaca is called a Cria e.d. CamelsAlpacas also are primarilyoriginate frombred forSouth their America fibre (wool)

Do you want to complete your CPD on the traditional • Brucellosis in South Africa – a notifiable medical condition webplatform or SMS system? Use the following codes - AC/2248/19 SMS code = a63262 for sms or follow the link from www.vet360.vetlink.co.za • The Importance of Teat Liners in Preserving Teat Integrity Some of the latest articles from Vetlink and Udder Health - AC/2186/19 SMS code = a23747 • Enzootic Abortion in Small Ruminants AC/2130/19 14 SMS code = a74310