world association for the advancement of veterinary parasitology (waavp): second edition of...

Veterinary Parasitology 103 (2002) 1–18

World association for the advancement of veterinaryparasitology (WAAVP): second edition of guidelinesfor evaluating the efficacy of equine anthelmintics

J.L. Duncana,∗, E.M. Abbottb, J.H. Arundelc,M. Eyskerd, T.R. Kleie, R.C. Krecekf , E.T. Lyonsg,

C. Reinemeyerh, J.O.D. Slocombeia Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK

b Hoechst Roussel Vet, Rheingaustrasse 190, Wiesbaden 65204, Germanyc 59 Illawarra Road, Hawthorn 3122, Victoria, Australia

d Division of Parasitology and Tropical Veterinary Medicine, Utrecht University,P.O. Box 80.165, 3508 TD Utrecht, The Netherlands

e School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USAf Department of Veterinary Tropical Diseases, Faculty of Veterinary Science,University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa

g Department of Veterinary Science, Agriculture Experimental Station,University of Kentucky, Lexington, KY 40546-00761, USA

h East Tennessee Clinical Research Inc., 4315 Papermill Road, Knoxville, Tennessee, TN 37922, USAi Department of Pathobiology, Ontario Veterinary College,

University of Guelph, Ont., Guelph, Canada NIG 2W1

Accepted 4 September 2001

Abstract

These guidelines have been designed to assist in the planning, operation and interpretation ofstudies which would serve to assess the efficacy of drugs against internal parasites of horses.Although the term anthelmintic is used in the title and text, these guidelines include studies ondrug efficacy against larvae of horse bot flies,Gasterophilusspp., which are non-helminth parasitescommonly occurring in the stomach of horses. The advantages, disadvantages and application ofcritical and controlled tests are presented. Information is also provided on selection of animals,housing, feed, dose titration, confirmatory and clinical trials, record keeping and necropsy proce-dures. These guidelines should assist both investigators and registration authorities in the evaluation

∗ Corresponding author. Tel.:+44-141-339-8855; fax:+44-141-942-7215.E-mail address:[email protected] (J.L. Duncan).

0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0304-4017(01)00574-X

2 J.L. Duncan et al. / Veterinary Parasitology 103 (2002) 1–18

of compounds using comparable and standard procedures with the minimum number of animals.© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Anthelmintic;Gasterophilusspp.; Horses; Guidelines

1. Introduction

Guidelines for evaluating the efficacy of anthelmintics in horses were first published in1988 (Duncan et al., 1988) and followed the publication of similar World Association forthe Advancement of Veterinary Parasitology (WAAVP) guidelines for testing anthelminticefficacy in ruminants (Powers et al., 1982) and swine (Düwel et al., 1986). Since then similarguidelines for dogs and cats (Jacobs et al., 1994) and revised guidelines for ruminants (Woodet al., 1995) have been published. This revision of the equine anthelmintic guidelines is afurther step towards the standardisation and refinement of methods for the evaluation ofanthelmintics in horses. When circumstances allow, the acceptance and use of the WAAVPanthelmintic guidelines by government authorities should be the basis for harmonisationand acceptance of trials performed in different countries.

The aim of the WAAVP is to establish uniform international standards for anthelminticevaluation to expedite the registration of effective agents. It is hoped that these detailedspecies guidelines will (1) serve as models for government officials responsible for deve-loping meaningful efficacy registration requirements within their countries, (2) assist inves-tigators in preparing basic plans to demonstrate effectively the efficacy of new anthelminticsand (3) which is important from the animal welfare viewpoint, reduce the number of exper-imental animals used for drug testing with obvious consequent reductions in overall costs.All studies should be conducted in accordance with existing local guidelines governinganimal experimental studies such as good target animal study practices (GTASP) in theUSA. It should be emphasised that appropriate biometric input is essential, especially atthe protocol development stage.

2. Guidelines

2.1. Methods of anthelmintic evaluation

Both the controlled and critical tests have been widely used and are acceptable in assessingthe efficacy of anthelmintics against many important parasitic helminths of horses. In mostcases, naturally infected animals may be used but it may be desirable in some cases to studydrug activity against a number of important species, such as some of the large strongyles,by experimental infections.

2.2. Controlled test

This is a reliable procedure for determining efficacy of anthelmintics in horses but becauseof the wide variation in numbers of parasites which may be present, a minimum of six but

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ideally 10 infected animals per group is recommended. Briefly, after an acclimation period,parasitised animals are randomly allocated into treated and untreated control groups and atest compound administered as appropriate. After about 2 or 6 weeks where effectivenessagainst immature stages is being considered, the animals are necropsied and the parasitesrecovered, identified and counted. The efficacy of the compound is then determined bycomparing the number of parasites in the controls with the number remaining in the treatedanimals. Percentage efficacy against each parasite species is determined by the followingformula:

percent efficacy=mean number of worms in control

−mean number of worms in treated animals

mean number of worms in controls× 100

Appropriate statistical methods should be used to determine the significance of efficacy(Appendix A).

2.3. Critical test

Although time-consuming and laborious this method may be used to assess efficacyagainst lumen dwelling gastrointestinal parasites using individual infected animals whichact as their own controls. The animals are maintained singly and dosed with a test compound.Faeces are collected daily from 1 day before to up to at least 7 days after administration ofthe drug when the animal is killed and necropsied. The parasites are collected from the dailyfaecal output and at necropsy those remaining in the gastrointestinal tract are recovered,identified and counted. The total of these collections is considered to be the worm burdenwhich existed at the time of treatment, and the efficacy against any particular species ineach individual animal is calculated from the following data:

percentage efficacy

= number of parasites expelled

number of parasites expelled+ number of parasites remaining at necropsy×100

A disadvantage of this test is that small nematodes normally found in the stomach or smallintestine may be partially digested when passed in the faeces and thus are difficult to findor identify. The use of the critical test for efficacy data against such parasites will thereforeresult in a conservative estimate of efficacy. Accuracy will also be affected by natural lossesof parasites during the faecal collection period. The pooled results from several criticaltests may give sufficient data for registration of a compound against individual parasitespecies.

Both controlled and critical tests may be used to assess efficacy against adult, and larvalstages where appropriate, for example in the case of larval stages present in the lumen ofthe gastrointestinal tract.

The controlled test, using natural or experimental infections, is suitable for determiningefficacy against larval stages which are not in the lumen of the intestine. In this case,


necropsy may be delayed to allow immature parasites in the controls and those which havesurvived treatment, to mature and thus facilitate collection and identification.

2.4. Selection of animals

Efficacy may be determined in either horses or ponies. Where possible, animals shouldbe obtained from the same source and be of similar breed/type and age. For most of theimportant parasite species, young (up to about 1–2 years old) naturally infected animals arelikely to have the highest numbers of a wide range of parasites and these should be selectedon the basis of faecal worm egg and/or differential larval counts. In any group, the animalsshould be from a similar parasitological background whether the infections have beenacquired naturally or experimentally induced. Where it is not possible to obtain naturallyinfected animals which have had similar background exposure, the group may be grazedtogether for a further 6–8 weeks on pastures known to be heavily contaminated with horseparasites prior to the commencement of a trial in an attempt to ensure adequate infections.Although rarely necessary in the horse, experimental infections may be superimposed onnaturally acquired infections to allow testing against as wide a range of parasite species aspossible. The animals, although parasitised, should be free of clinical signs of any otherinfections. All animals should be weighed both prior to the start and at the end of thestudy. This will aid randomisation to treatment and control groups. Test animals should,in addition, be weighed 24 h prior to treatment in order to calculate accurately individualdoses of the specific drug formulation to be administered.

2.5. Allocation of animals

In the case of controlled tests, the animals should be randomly allocated to treated andcontrol groups. Replicates of animals with similar characteristics (liveweight/faecal eggcounts, etc.) are formed and within each replicate animals are allocated at random to treatedor control group.

2.6. Housing

All animals used in a controlled test should, whether naturally or experimentally infected,be housed or confined under similar conditions and these should be designed to minimiseexposure to parasites. In general, housing should take place at least 7 days prior to experi-mental infection or treatment to allow acclimatisation to the surroundings and feed.

Horses used in critical tests should also be housed 7 days prior to infection or treatmentbut they should be maintained individually under conditions which allow daily collectionof faeces from each animal. Faeces should be collected and screened for grossly visibleparasites and samples examined for small worms on the day prior to treatment to determinewhether the change in housing or feeding is causing spontaneous parasite elimination. Anyanimals found to be passing large numbers of parasites prior to treatment should be excludedfrom the experiment.

Stalls or pens should be so constructed that manure can be removed daily and theaccommodation thoroughly cleaned.


2.7. Feed

In general, the selection of the ration is at the discretion of the investigator but it shouldbe a commonly used feed, nutritionally adequate and the same for all animals, and shouldnot be changed during the acclimatisation period or during the course of the trial. An-thelmintic formulations can be mixed with the animal’s normal ration or the drug may beincorporated, at the manufacturing stage, in a proprietary pelleted feed. For the latter, ananalysis of the feed including drug concentration should be provided by the sponsor priorto the study start and retention samples kept from each batch manufactured until the trial iscompleted.

If a drug has to be administered in a special ration, this should be fed during the 7-dayacclimatisation period to allow the animals to become accustomed to the new diet. If amedicated pelleted feed is to be used, all animals should be offered unmedicated pelletsduring the acclimatisation/treatment period in the same proportion as the medicated pelletswill be offered to the treatment group. The untreated animals will then serve as placebocontrols. Whichever method of in-feed administration is used, the drug should be admin-istered, according to manufacturer’s instructions, in a small quantity of the ration beforeoffering the remainder of the unmedicated feed. Animals need to be individually pennedduring administration of the feed to ensure correct dose intake. A record should be keptof the time taken for each animal to consume the feed. If the medicated feed is not con-sumed within a reasonable period of time (usually 18–24 h), then the weight of the amountremaining should be recorded because data from this animal are unlikely to be acceptablein support of an efficacy claim. To avoid this, palatability trials should be conducted beforeefficacy trials to ensure that animals will consume the medicated feed. If possible, it is agood practice to record daily feed intake of both treated and control animals before andduring the trials. The time and extent of any loss of appetite should be noted.

At the conclusion of a trial, feed may be withheld for 24 h prior to necropsy to facilitateparasite recovery and counting. If bodyweights are recorded post-treatment, these shouldbe taken before withdrawal of feed.

3. Efficacy against adult worms and lumen-dwelling larvae of themajor equine parasites (see Table 1)

Controlled and/or critical tests may be used to assess efficacy against migratory(Strongylusspp.) and other non-migratory (Triodontophorusspp.,OesophagodontusandCraterostomum) large strongyles, small strongyles (Cyathostominae), ascarids (Parascarisequorum), pinworms (Oxyuris equi), tapeworms (Anoplocephalaspp. and other genera)and larvae of horse bot flies (Gasterophilusspp.).

3.1. Large strongyles

There are three species of large strongyles, namelyStrongylus vulgaris, Strongylusequinusand Strongylus edentatus, that undergo extensive migrations as parasitic larvaein various tissues of the host. They require approximately 6–12 months after infection to

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become mature adults in the intestine; thus, naturally infected animals over 1 year of agemust be used to test drug efficacy against adult parasites of these three species.

3.2. Small strongyles

There are over 40 species of small strongyles whose normal maturation periods rangefrom 6 to12 weeks but this can be extended to several years if development is inhibitedor arrested at the early third larval stage (EL3). A variable period of larval developmenttakes place within the large intestinal mucosa before fourth stage larvae emerge into the gutlumen. Untreated animals under field conditions acquire large burdens of small strongylesthroughout life and, although animals over several months of age are suitable for drugtesting, yearling or 2 year old animals are preferable so that efficacy against the slowermaturing large strongyles can also be determined.

3.3. Ascarids

The highest numbers of horse ascarids are found in grazing animals of 3–18 months ofage and animals from this age group should be selected, on the basis of high faecal eggcounts, for drug testing againstP. equorum.

3.4. Pinworms

Five months are required for maturation ofO. equi, and adult worms are most commonlyfound in older foals and yearlings that have been stabled. There is a short period of develop-ment within the intestinal mucosa before fourth stage larvae emerge into the lumen of the gut.Large numbers of larvae are often present without mature worms, especially in older animals.

3.5. Tapeworms

Tapeworm (Anoplocephalidae) infections of horses are acquired during grazing by in-gestion of forage (oribatid) mites containing the cysticercoid stage. After 1–2 months adultworms are found in the small intestine and caecum and mature segments and eggs are passedin the faeces. Horses of all ages may be infected and there appear to be slight seasonal fluc-tuations in parasite numbers in temperate areas. Mite numbers reach a maximum in springand early summer and ingestion of infected mites during this period leads to the heaviestburdens of tapeworms being found from mid-summer to autumn. There is then a gradual lossof worms and lowest numbers are usually found in the spring. There are special difficultiesassociated with assessing drug efficacy against tapeworms in the horse and to overcomethese Lyons and Slocombe have suggested modifications of the critical test (Appendix A).

3.6. Bots

Eggs of the horse bot fliesGasterophilusspp. are laid on the hairs of the horse during thesummer months in temperate areas of the world. Hatched larvae migrate from the mouth tothe stomach where they spend approximately 10 months before being passed in the faeces.


Infection occurs in animals of all ages but, as they start to be passed in the faeces, thenumbers of mature (third instar) larvae decline in the spring to low levels in summer intemperate regions, so these periods should be avoided when planning a drug trial againstbots. In warmer areas, bots at various stages of development can be found in the stomachthroughout the year.

In a critical test, nearly all of the above gut lumen-dwelling parasites are found during the2nd–4th day following treatment but faeces should be examined for about 6–7 days whenthe animals will be necropsied. An interval of 7 days between treatment and necropsy isusually suitable in controlled tests, but a longer interval may be used under some circum-stances, e.g. 2 weeks is considered the minimum interval for examination for the eliminationof bots.

It is probably simpler to use naturally infected animals for drug testing against the aboveparasites whether using critical or controlled tests since the establishment of experimentalinfections in animals reared worm-free is a time consuming and extremely expensive pro-cedure and is probably warranted only where suitably infected animals cannot be identifiedor if persistent activity against helminths is to be examined.

4. Efficacy against tissue-dwelling larval stages of the major equine parasites

A controlled test on suitably infected animals is adequate to determine efficacy of acompound against tissue-dwelling larval stages of the major gastrointestinal helminths.Infections may be artificially induced or naturally acquired and it may be necessary tosubmit data from several independent trials in support of a claim for larvicidal activity. Themost important groups of parasites in this respect are the large and small strongyles.

4.1. Large strongyles

In the case of the large strongyles,S. vulgaris andS. edentatus, the predilection sitesshould be examined at necropsy for developing larvae; these are the cranial mesentericartery and retroperitoneal tissues of the abdominal wall, respectively. In the case ofS. equinus, the migratory phase of the life-cycle involves the liver and the pancreas andthese organs should be examined for such larval stages.

4.2. Small strongyles

Third and fourth stage larvae of the small strongyles are to be found developing in the largeintestinal mucosa, and samples of these tissues obtained at necropsy should be examinedand the numbers of larvae and, if possible, the stage of development recorded. The verysmall early L3 can only be found after peptic digestion of these tissues whereas the largerdeveloping L3 and L4 are visible and can be enummerated using transillumination of themucosa using a strong light source. The latter can also be recovered following digestionthough some may be lost during the process. Despite these losses many laboratories usepeptic digestion only as the technique is more efficient and less time consuming (for methodssee Appendix A).


4.3. Ascarids

Recovery of early larval stages ofP. equorummigrating through the liver and lungs isextremely difficult. To demonstrate efficacy against these stages, experimental infectionsshould be used followed by treatment 1–2 weeks later and subsequent comparison of thenumbers of worms which have returned to the small intestine in treated and untreatedcontrols. Necropsy examination 4–6 weeks after infection is usually suitable.

5. Titration of the recommended dose

Dose titration/determination trials are usually carried out using controlled tests and willnormally follow preliminary ‘in house’ studies which have indicated the potential spectrumof activity and the approximate dose. If broad-spectrum activity is anticipated then animalsunder test should be infected with at least several of the major groups of equine parasites.Trials should be carried out with the final or near-final formulation and the range of dosagerates selected on the basis of preliminary studies which have indicated the approximateeffective dosage rate.

Treated and non-treated control groups should consist of sufficient animals infected withadequate numbers of each parasite species or group of species, e.g. the small strongyles,to provide evidence of significant anthelmintic efficiency within the range of dosage rates.The larger the number of animals and the more uniform the pre-treatment burdens of thevarious parasites, the more accurately anthelmintic activity may be demonstrated.

Included in the trial there should normally be a minimum of three groups receivingdifferent levels of treatment together with a group of untreated controls. Of the treatedanimals, individuals in one group should be treated with the anticipated recommended doseof the final formulation, while those of the second and third groups should be treated witha dose lower and higher, respectively, than the anticipated dose. Occasionally it may beacceptable to some regulatory authorities to include only two treatment groups in dosetitration trials if there is good evidence from preliminary studies of high efficacy. Theefficacies of each dosage rate are determined by comparison of the worm burdens in thetreated and control animals at necropsy. The route of administration and the formulation ofthe product should be those proposed for marketing, with the exception that the amount ofactive ingredient might be altered in concentration.

Data obtained in a dose titration trial may be acceptable as one of the dose confirmationtrials if the compound used was in its final or near-final formulation and was administeredas recommended according to label directions; also, the worm burdens of individual speciesor groups for which a claim is to be made must have been adequate.

6. Protocols for proposed dose confirmation and clinical trials

On completion of the dose titration studies it is recommended that proposed dose confir-mation and clinical trials, which are required to substantiate claims against various speciesof parasites, are conducted in accordance with the relevant local registration requirements.


For example it is a requirement of some registration authorities to review study protocolsprior to the start of the study. Guidelines on protocol preparation are provided by FEDESAin the EU and by the FDA in the USA.

Protocols should provide information on the number, breed and age of animals to be usedand whether these will be artificially or naturally infected. They should also indicate thespecies and stages of infection against which the drug will be evaluated and the methodswhich will be employed to recover and enumerate both adult and immature parasites.

Other pertinent information should include how efficacy is to be measured, including sta-tistical analysis, the proposed experimental design and the qualifications of the investigators.

6.1. Dose confirmation trials

These trials should be conducted using the final or near-final formulation, dosage rateand route of administration proposed for marketing. The product must have the effect to beclaimed on the proposed label when used as recommended. At least two dose confirmationtrials following identical protocols will be required to demonstrate efficacy of a new productagainst a particular species or, where relevant, a group of related species such as the smallstrongyles, which will appear on the label. Although not a universal requirement, in somecountries the species of small strongyles must be identified. These trials should be carriedout at different locations and one should be conducted by an investigator independent ofthe sponsor. It is required that at least one trial for each species or group of species claimedshould be conducted with naturally infected animals. To avoid bias the studies should beconducted so that personnel involved in data collection are “blind” as to treatment and thata randomised study design is used.

To determine the efficacy of a compound against a single parasite species or a group ofspecies in both dose titration and dose confirmation trials, a minimum of six adequatelyinfected horses per group is required in each trial. Although six animals with adequatenumbers of a particular parasite species may be acceptable, more will probably be requiredin order to obtain adequate numbers of all the species to be claimed and to demonstratestatistically significant difference in parasite burdens between treated and control animals.

It is generally not acceptable to compile data on individual species from separate trialscarried out by different investigators in different areas, in order to satisfy minimum datarequired from the two dose confirmation trials.

The appropriate dose of the drug, as established in the dose titration trial, is administeredto each animal in accordance with the proposed label instructions and, if medicated feed isused, daily feed consumption should be recorded to verify that all such feed is consumedby each animal.

A claim for efficacy against immature parasites should refer to the stage of development(in the case of natural infections) or to the age of the parasite in days (in the case ofexperimental infections) at the time of treatment.

Although label claims for efficacy against the major horse parasites should generallybe for genus, species and stage of infection, with the exception of a few regulatory au-thorities, it may be acceptable to include data for the small strongyles and non-migratorylarge strongyles as a group. The small strongyles, now commonly referred to as cyatho-stomes, include the following common genera:Cyathostomum, Cylicodontophorus,


Cylicocyclus, Cylicostephanus, Poteriostomum, Gyalocephalus, Caballonema andCylindropharynx. Non-migratory genera of the large strongyles such asTriodontophorus,OesophagodontusandCraterostomumare often included with the small strongyles for thepurposes of anthelmintic efficacy testing since their behaviour and location within the hostare similar.

For a recent description of the genera and species of equine helminths see Lichtenfelset al. (1998) and for details of the evaluation of anti-parasitic drugs in horses the reader isreferred to the publication of Drudge and Lyons (1977).

Label claims should be made only for the specific parasites against which the compoundwas tested and proved effective. In such claims, a mean percentage reduction in worm num-bers of 90% or more is normally considered effective if a statistically significant differencein parasite numbers exists between control and treated animals.

6.2. Clinical trials

Clinical trials are conducted primarily to evaluate further the performance of the productas used by the consumer in the field and to extend experience on the safety of the drug whenit is used in different breeds and ages of horses. Efficacy should be determined in a numberof breeds or types of animals in several different geographical locations because of variableenvironmental conditions, possible strain variations of parasite populations, including drugresistant forms, and different feeding and management practices. Animals in the treatmentgroup should receive the recommended dose of the final formulation of the product to bemarketed.

In this type of trial, antiparasitic efficacy is almost always determined by faecal egg countsand in some instances by larval counts and larval culture/differentiation. Faecal samplesshould be examined from each animal at the time of treatment and again at 7 and 14 daysafter therapy. It is recommended that faecal egg counts are monitored at intervals for longerperiods (6 weeks–6 months) to provide information on likely treatment intervals. Adequateuntreated controls, e.g. one for every four treated animals, should be included in the trial.With the exception of treatment, these animals should be handled in the same manner asthe treated animals. If possible, data from at least 100 treated animals should be obtainedin each of three different areas. Fewer animals may be included in each area if more thanthree different areas are used, but data from an aggregate total of at least 300 treated horsesare recommended. Any of the commonly used egg/larval counting techniques is acceptablebut the same method should be used at all trial sites. The technique used should be reportedalong with the other data.

Again, as with the dose confirmation studies bias should be prevented by the randomselection of treated and untreated animals and by the ‘blind’ evaluation of results. Forexample, the individuals performing faecal examinations should not collect the samplesand should not be aware of the treatment status of the animals from which the samples weretaken. Any other or additional means of excluding or minimising bias may be employed.

The clinical investigator must record any side effects observed after therapy. Animalsshould be observed routinely for several hours after treatment and the owner or otherresponsible person should be advised to observe the animals at specified times and reportany reaction seen or suspected during the trial.


Any animals that die during dose titration, dose confirmation or clinical trials must benecropsied and complete records submitted.

6.3. Trials to assess persistent efficacy

Some modern anthelmintics show persistent effect not only against the establishmentof new infections but probably also against reactivated tissue dwelling larval stages. Totest for the latter, however, is almost impossible, but prolonged egg reappearance times inanimals treated and subsequently kept under conditions designed to prevent reinfection,might indicate persistent drug efficacy.

Any trials designed to confirm persistent activity against new infections with equinehelminths would require different groups of helminth-naı̈ve animals to be treated and sub-sequently exposed to single or multiple experimental or natural infections at varying periodspost-treatment. Claims for efficacy would be determined by a comparison of worm countsat necropsy (Vercruysse et al., 1998).

Any protocols designed to determine persistent drug effects against different species ofequine helminths should be discussed in detail with the appropriate regulatory authoritiesbefore studies are initiated.

6.4. Change of formulation

If the sponsor desires to change the formulation, dose regimen or route of administrationof an approved product, bioequivalency studies, which may include comparative necropsytrials, will be required to demonstrate that the alteration has not resulted in a change ofefficacy compared with the approved product. At least one dose confirmation trial in animalsinfected with adequate infections of the least drug-sensitive parasites claimed on the labelwill probably be necessary for demonstrating comparability. In certain instances, additionaldose titration and/or clinical trials may be necessary but these will usually be conductedwith fewer animals than the number required for initial approval.

6.5. Records and reports

Complete records should be kept of the experimental procedures used in an anthelminticefficacy investigation. These records should include items such as (1) number of experimen-tal animals, condition and allocation of animals; (2) source, breed, age, weight, and sex ofanimals; (3) housing, feed, number and source of infective larvae if experimental infectionsare used; (4) dose rate, dose route, time of treatment, adverse reactions, lot number, date ofreceipt, formulation and storage conditions of the test compound; (5) necropsy procedures,gross pathological changes, identification and counts of parasites recovered by direct countsor aliquots; (6) any other information, such as statistical analysis, disposal of carcases anddaily health records which is pertinent to the experiment. Individual records for each animalin a study are of value and should contain all important events and dates of these events.Copies of original raw data should accompany the report.

A summary sheet of the pertinent efficacy data should be prepared for each investi-gation and should contain the following data for each animal in the study: the actual


amount of active ingredient administered; number, frequency and volume of doses; thestages of infection; the number of parasites recovered from each animal in the faeces andat necropsy according to species and location with the mean numbers in control and treatedgroups; the percentage removal for each parasite in critical or controlled tests and the meannumbers of eggs and/or larvae for the control and treated groups in clinical trials. A sum-mary statement and conclusions of the work performed should be prepared and submittedby each investigator.

Acknowledgements

Sincere gratitude is due to the six leading international experts who kindly reviewed thisdocument, the final version of which was approved by the Executive Committee of theWAAVP.

Appendix A. Necropsy and parasitological techniques

In critical tests, necropsy is usually performed 6–7 days after treatment to estimate wormburdens present compared with numbers of parasites expelled in the faeces. In controlledtests necropsies may be carried out between 1 and 2 weeks post- treatment to assess efficacyagainst gut lumen-dwelling adults and larval stages and migratory stages of certain parasitespecies, for exampleS. vulgarisin the cranial mesenteric arteries orS. edentatusin theretroperitoneal tissues of the abdominal cavity.

Necropsy may be delayed for 4–6 weeks especially to establish efficacy against early thirdstage larval (EL3) cyathostome larvae inhibited in their development in the large intestinalmucosa. By delaying necropsy for this period and ensuring that the experimental animalsare not exposed to re-infection after the treatment date, it can be assumed that any EL3 foundare definitely inhibited in their development. This will allow a more accurate assessmentof efficacy against EL3. Disadvantages of delayed necropsy are that natural eliminationof parasites from the controls during an extended post-treatment period may lead to anunderestimate of efficacy and that development of EL3 to late third stage larvae or fourthstage larvae (LL3/L4) will not allow efficacy against these to be established accurately.Earlier necropsy at 1–2 weeks may be more appropriate for assessment of efficacy againstLL3/L4. At present there is no method available at necropsy which will differentiate betweenLL3/L4 stages which are undergoing normal development from these which may be inhibitedat these stages of development.

As soon after death as possible the gastrointestinal tract should be removed in its entiretyand the various segments isolated and separated by double ligatures. Each segment shouldthen be opened, the contents removed and the mucosa thoroughly washed to remove freeparasites.

The contents and washings from each segment are then combined. For evaluation ofactivity against small species, aliquots are then taken before the entire contents of the seg-ment are examined for grossly visible large parasites such asP. equorum, large strongylesor bots. In the case of the large intestine it is now generally considered that the contentsand the washings should not be combined and that samples from both should be


examined separately. This will allow a better estimation of less numerous large speciessuch asStrongylusspp. orTriodontophorusspp. which remain attached to the mucosa forvarying periods after death: these are removed and added to the washings.

For evaluation of activity against small parasites, aliquot samples are taken from thecontents of each gut segment and examined microscopically or under a magnifying lens.Examination of samples and counting of parasites may be facilitated by washing the aliquotsfirst through a fine sieve of suitable aperture mesh to remove large parasites and particulatematerial which will result in a clearer background and facilitate microscopical examinationfor small parasites.

A.1. Stomach

Once the stomach contents and washings have been collected, bot larvae that haveremained attached during the washing may be picked off. Bots recovered from both themucosa and contents are identified and counted.

The mucosa of the stomach should then be subjected to digestion by standard pepsin/HCltechniques used in most parasitological laboratories. Alternatively, the mucosa can besoaked in water or saline overnight at room temperature or incubated at 37◦C for 5 hours.These methods are used in order to recover anyHabronema, Draschiaor T. axeilocatedwithin the mucosa, and aliquots of the digest are examined to enumerate these parasites.Granulomatous lesions provoked by infection withDraschiashould be opened and exam-ined for worms.

A.2. Small intestine

The majority ofParascariscan be collected as the small intestine is opened. All of thispopulation is identified and counted as are all tapeworms with intact scolices. The smallintestinal contents may be examined for larvalP. equorumandS. westeri;digestion of themucosa of the duodenum and proximal jejunum facilitates the recovery ofS. westeri. Fordemonstration of anthelmintic activity againstS. westeriit will be necessary to use foalsless than 5 months of age.

A.3. Caecum and colon

The contents of the caecum, ventral colon and dorsal colon from individual animals areeach mixed uniformly and 10% aliquots taken. The total contents and washings of the threeseparate parts of the large intestine, should be carefully examined for any large parasitespresent. Aliquots of the washings may be examined microscopically for small parasites.

The majority of adult and small strongyle L4, and immatureO. equiare usually foundin aliquots of the contents. When aliquots are used, all parasites in the aliquots should beidentified and counted. If the worms are extremely numerous then it may be necessary toprepare subaliquots for counting. The total numbers of each parasite recovered from thefaeces and at necropsy are then calculated.

Uniform samples of tissues are examined for encysted small strongyle larvae in themucosa. Two techniques, digestion and transmural illumination have been used, but the


current consensus is that digestion is the more reliable; for this, pepsin/HCl digestion ofknown weights of mucosa releases larvae for enumeration. This, however, may result inlarval damage/digestion with a consequent artificially low rate of larval recovery, especiallyof LL3/L4. Direct examination of uniform areas of gut wall under magnification may bepreferred and counting is facilitated by provision of a strong light source under the sampleand by excision of the taeniae or bands of the caecum and colon which will overcomethe problem of extensive folding of the large intestinal mucosa. Disadvantages of thistechnique are that it cannot detect very small EL3 and that it does not allow differentiationbetween dead and viable LL3/L4. If both techniques are applied to the same samples, that istransmural illumination (for LL3/L4) followed by peptic digestion (for EL3), this is likelyto give the most accurate counts of cyathostome larvae encysted in the large intestinalmucosa. Samples are obtained by cutting 20 cm2 longitudinal strips or squares of gut wallalong the length of each segment between the taenial bands. The combined weight ofthese samples should be approximately 5–10% of the total weight of the washed caecum,ventral colon or dorsal colon. The total weight of each of these three segments of the largeintestine divided by the combined weight of the pieces removed from each segment isthe multiplication factor applied to calculate the numbers of encysted cyathostome larvaepresent in the large intestinal mucosa. Although minor modifications are applied in somelaboratories, the following are the standard techniques usually employed.

A.4. Transmural illumination

On the day of necropsy each sample of excised tissue is stretched, after removal ofthe serosa with scissors, on a calibrated petri dish. The samples are then examined, usinga dissecting microscope at 10–15X magnification, and the encysted cyathostome larvaecounted. The total number of LL3/L4 in each segment is then calculated using the appropriatemultiplication factor.

A.5. Mucosal digestion

The mucosa from each sample is scraped off with a microscope slide and the combinedmaterial from all samples digested in a freshly prepared pepsin/HCl solution (10 g, 1:10,000pepsin and 15 ml concentrated HCl in l l distilled water) by incubation at 37–40◦C for aminimum of 2 h. The samples are frequently shaken or placed in a heated shaker waterbath to aid digestion and as a rough guide 200 ml digestion fluid is added to approximately30 g mucosal tissue. Digestion should continue until no obvious large pieces of tissueare visible and it may be beneficial to remove the supernatant and add fresh pepsin/HClsolution after each 2 h incubation period. The digested samples from each segment arebulked and sedimented to a final volume of 400–500 ml and formal saline may then beadded to allow subsequent examination. To aid examination the digestion mixture maybe washed through a sieve (mesh aperture 63�m) and the retained material reconstitutedto 400–500 ml with water. Suitable aliquots are then stained with iodine and examined at20X magnification using a dissecting microscope. The total number of EL3 estimated to bepresent in the combined mucosal samples is then multiplied by the appropriate multiplicationfactor to provide an estimate of the total number of EL3 present in each segment of the


large intestine. Although further differentiation of mucosal larval stages has been reported,their enumeration as mucosal EL3 and mucosal LL3/L4 present at necropsy 4–6 weeksafter treatment in a controlled trial probably provides the clearest and best estimate ofanthelmintic efficacy. For a review of mucosal cyathostome larval recovery techniques seeEysker and Klei (1999).

A.6. Statistical analysis

Appropriate statistical methods should be used to analyse the data. These should bedocumented giving details of methodology, references and calculations. Any questionsregarding the selected procedures should have been discussed with statisticians of theappropriate authority in the early stages of protocol development.

In general, it is suggested that both a parametric and a non-parametric test be carried out.If both are significant or non-significant, the efficacy or lack thereof is proved beyond anyreasonable doubt.

A.7. Testing against individual parasite species

Table 1 presents a list of the gastrointestinal parasites of the horse with an indicationof which test and type of infection is suitable. From this, it is clear that it is possible togenerate efficacy data for all of the parasites listed using the controlled test in naturallyinfected animals. However, if suitable infected animals are difficult to obtain, it may provemore expedient to carry out efficacy studies in animals with experimentally induced infec-tions. Such studies have been used to assess drug efficacy againstGasterophilusspp. larvae,T. axei, P. equorum, S. westeri, Strongylusspp. and small strongyle larvae (Drudge et al.,1963, 1972, 1982; Duncan et al., 1977; Lyons et al., 1982, 1985; Malan et al., 1981a,b;Slocombe et al., 1982).

A.8. Testing against tapeworms

The method recommended for specific testing againstAnoplocephalaspp. is based on amodified critical test described by Lyons et al. (1989) with further modifications suggestedby Slocombe (personal communication). This involves necropsy of animals 48 h after treat-ment with collection and examination of all faeces passed during this period for tapeworms.Evaluation of efficacy is based on counts of tapeworms found in faeces compared with thosepresent in the small and large intestine at necropsy. NormallyAnoplocephalaspp. are foundmainly in the ileum and caecum and specimens found in the small intestine and caecum atnecropsy are considered to have been unaffected by treatment; until there is more evidenceto the contrary those found unattached in the ventral colon, dorsal colon, small colon andrectum should be considered to have been removed by drug treatment.

A.9. Testing against Dictyocaulus arnfieldi

There have been few published reports of anthelmintic efficacy againstD. arnfieldi inhorses. This is due largely to the fact that lungworm infections in horses rarely develop


to patency, and diagnosis is frequently based on clinical signs of chronic coughing anda history of contact with donkeys, which are generally considered to be the natural hostsof D. arnfieldi. Until recently, recommendations for anthelmintic treatment against theequine lungworm have been based on the clinical response to treatment, faecal larval countspre- and post-treatment and extrapolation of the results of efficacy studies in donkeys inwhich patent infections are readily found. For methods of evaluating drug efficacy againstD. arnfieldi in donkeys and horses it is suggested that the investigator refers to the publi-cations by Clayton and Neave (1979), Britt and Preston (1985), and Lyons et al. (1985).

A.10. Testing against other equine parasites

These guidelines are unable to offer detailed recommendations for the testing ofanthelmintic efficacy against extra-intestinal parasites such asThelazia, Onchocerca, Para-filaria andHabronemaorDraschialarvae in cutaneous lesions. Protocols for trials to assessefficacy against such parasites should be discussed with appropriate regulatory authoritiesprior to the commencement of any studies.

References

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Clayton, H.M., Neave, R.M.S., 1979. Efficacy of mebendazole againstDictyocaulus arnfieldiin the donkey. Vet.Rec. 104, 571–572.

Drudge, J.H., Lyons, E.T., 1977. Methods in the evaluation of antiparasitic drugs in the horse. Am. J. Vet. Res. 38,1581–1586.

Drudge, J.H., Lyons, E.T., Swerczek, T.W., 1972. Activity of dichlorvos against first instars ofGasterophilusspp.Am. J. Vet. Res. 33, 2191–2193.

Drudge, J.H., Lyons, E.T., Tolliver, S.C., 1982. Controlled tests of pastes of dichlorvos and thiabendazole againstinducedStrongyloides westeriinfections in pony foals in 1973–1974. Am. J. Vet. Res. 43, 1675–1677.

Drudge, J.H., Szanto, J., Wyant, Z.N., Elam, G., 1963. Critical tests of thiabendazole as an anthelmintic in thehorse. Am. J. Vet. Res. 24, 1217–1222.

Duncan, J.L., McBeath, D.G., Best, J.M.J., Preston, N.K., 1977. The efficacy of fenbendazole in the control ofimmature strongyle infections in ponies. Equine Vet. J. 9, 146–149.

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Düwel, D., Barth, D.W., Batte, E.G., Berger, H., Stewart, T.B., Theodorides, V.J., 1986. World Association for theAdvancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anthelmintics inswine. Vet. Parasitol. 21, 69–82.

Eysker, M., Klei, T.R., 1999. Mucosal larval recovery techniques of cyathostomes: can they be standardized? Vet.Parasitol. 85, 137–149.

Jacobs, D.E., Arakawa, A., Courtney, C.H., Gemmell, M.A., McCall, J.W., Myers, G.H., Vanparijs, O., 1994.World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating theefficacy of anthelmintics for dogs and cats. Vet. Parasitol. 52, 179–202.

Lichtenfels, J.R., Kharchenko, V.A., Krecek, R.C., Gibbons, L.M., 1998. An annotated checklist by genus andspecies of 93 species level names for 51 recognized species of small strongyles (Nematoda:Strongyloidae:Cyathostominae) of horses, asses and zebras of the world. Vet. Parasitol. 79, 65–79.

Lyons, E.T., Drudge, J.H., Tolliver, S.C., 1982. Ivermectin: Activity against larvalStrongylus vulgarisand adultTrichostrongylus axeiin experimental infections in ponies. Am. J. Vet. Res. 43, 1449–1450.


Lyons, E.T., Drudge, J.H., Tolliver, S.C., 1985. Ivermectin: treating for naturally occurring infections of lungwormsand stomach worms in equids. Vet. Med./S.A. Clin. 80, 58–64.

Lyons, E.T., Drudge, J.H., Tolliver, S.C., Swerczek, T.W., Collins, S.S., 1989. Determination of the efficacy ofpyrantel pamoate at the therapeutic dose rate against the tapewormAnoplocephala perfoliatain equids usinga modification of the critical test method. Vet. Parasitol. 31, 13–18.

Malan, F.S., Reineke, R.K., Scialdo Rosina, C., 1981a. Recovery of helminths post-mortem from equines. I.Parasites in arteries, subperitoneum, liver and lungs. Onderstepoort J. Vet. Res. 48, 141–143.

Malan, F.S., Reineke, R.K., Scialdo Rosina, C., 1981b. Recovery of helminths post-mortem from equines. II.Helminths and larvae ofGasterophilusin the gastrointestinal tract and oestrids from the sinuses. OnderstepoortJ. Vet. Res. 48, 145–147.

Powers, K.G., Wood, I.B., Eckert, J., Gibson, T., Smith, H.J., 1982. World Association for the Advancement ofVeterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anthelmintics in ruminants (bovineand ovine). Vet. Parasitol. 10, 265–284.

Slocombe, J.O.D., McCraw, B.M., Pennock, P.W., Vasey, J., 1982. Effectiveness of ivermectin against late fourthstageStrongylus vulgarisin ponies. Am. J. Vet. Res. 42, 1526–1529.

Vercruysse, J., Eysker, M., Demeulenaere, D., Smets, K., Dorny, P., 1998. Persistence of the efficacy of a moxidectingel on the establishment of cyathostomes in horses. Vet. Rec. 143, 307–309.

Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, T., Malone Jr., J.B., Pankavich, J.A., Reinecke,R.K., Taylor, S.M., Vercruysse, J., 1995. World Association for the Advancement of Veterinary Parasitology(WAAVP) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine,caprine). Vet. Parasitol. 58, 181–213.

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