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Parasitol Res (2012) 111:2123–2127 DOI 10.1007/s00436-012-3060-1 ORIGINAL PAPER Treatment of third-stage larvae of Toxocara cati with milbemycin oxime plus praziquantel tablets and emodepside plus praziquantel spot-on formulation in experimentally infected cats Sonja Wolken & Claudia Böhm & Roland Schaper & Thomas Schnieder Received: 13 July 2012 / Accepted: 17 July 2012 / Published online: 4 August 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abs
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  ORIGINAL PAPER  Treatment of third-stage larvae of  Toxocara cati  with milbemycinoxime plus praziquantel tablets and emodepside plus praziquantelspot-on formulation in experimentally infected cats Sonja Wolken & Claudia Böhm & Roland Schaper & Thomas Schnieder Received: 13 July 2012 /Accepted: 17 July 2012 /Published online: 4 August 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Toxocara cati is the most prevalent gastrointesti-nal helminth in cats worldwide, with cats of all ages at risk of infection. An anthelminthic treatment that not only affectsthe gut-dwelling stages of this parasite but is also effectiveagainst developmental stages in the tissue has the advantagethat the pathology caused by migrating larvae is minimizedand the need for repeated treatments is reduced. This studywas conducted to evaluate the efficacy of milbemycin ox-ime/praziquantel tablets (Milbemax®, Novartis) against third-stage larvae of  T. cati in comparison to a spot-onformulation of emodepside and praziquantel (Profender®,Bayer). Twenty-four kittens were experimentally infectedwith T. cati and randomly allocated to three study groups.Treatments were performed at the minimum therapeuticdosage 5 days after the experimental infection. The devel-opment of patent infections was monitored and all cats weredewormed 50 days post-infection. Efficacies were calculat-ed based on counts of excreted worms in the treated groupscompared to a negative control group. Seven of the eight cats in the negative control group developed a patent  T. cati infection and all cats were excreting worms at the end of thestudy (geometric mean worm count 18.1). No efficacy could be observed for the milbemycin oxime-treated animals. Allcats developed a patent infection and excreted worms (geo-metric mean worm count 27.7). The treatment withProfender® was 98.5 % effective against L3 of  T. cati .One cat developed a patent infection and was excretingworms at the end of the study (geometric mean worm count 0.3). No adverse reactions were noted in either treatment group. Introduction Despite the availability of numerous highly efficaciousanthelminthic products, worm infestations remain the most common finding in companion animals, with Toxocara cati  being the most prevalent gastrointestinal helminth in catsworldwide. Analysis of lab data (Barutzki and Schaper 2003,2011) in Germany showed that no significant changes in prevalence rates have been observed over the last 12 years. These results demonstrate the constantly high needfor further efforts to control these pests and once againillustrate the parasites' effective survival strategies.The high reproductive rate of ascarids and the extremelyhigh tenacity of the eggs are major contributors to their success story. This indicates that strategic control of these parasites should minimize the contamination of the environ-ment by animals that harbour patent infections. Besides thisepidemiological aspect, it is also beneficial for the individ-ual patient to be cleared of an existing infection as soon as possible to minimize the pathology caused by migratinglarvae. In 1956, Sprent described the tracheal migrationroute of  T. cati after egg ingestion and in this context observed the occurrence of haemorrhagic spots on the lungs.Later, it was shown that the pathological changes caused bymigrating T. cati larvae include proliferation of the intima and media, which may result in complete occlusion of the pulmonary arteries (Swerczek et al.1970; Weatherley andHamilton1984). S. Wolken : C. Böhm ( * ) : T. Schnieder Institute for Parasitology, University of Veterinary MedicineHannover, Foundation,Buenteweg 17,30559 Hannover, Germanye-mail: Claudia.Boehm@tiho-hannover.deR. Schaper Bayer Animal Health GmbH,51368 Leverkusen, GermanyParasitol Res (2012) 111:2123  –  2127DOI 10.1007/s00436-012-3060-1  Since then, little attention has been paid to the pathologycaused by early-migrating T. cati larvae, and currently, effi-cacy against third-stage larvae (L3) has been demonstratedonly by one product (Profender®) (Reinemeyer et al.2005).Profender® is thus the only product licenced for the treat-ment of these early stages of  T. cati . Recently, the role that  parasites plays in the development of acute and chronic lungdisease in cats has been reinvestigated, and importance has been attached not only to the more obvious lung-relevant  parasiteslike  Dirofilariaimmitis and  Aelurostrongylusabstru- sus but also to the migrating stages of  T. cati (Dillon2011).This study aimed to evaluate and compare the efficacy of emodepside and the macrocyclic lactone milbemycin oximeagainst  T. cati L3. For the latter, efficacy against gut-dwelling fourth-stage larvae has been demonstrated(Schenker et al.2007), but no information is available on theefficacy against the stage of  T. cati which affects the lungs. Materials and methods The study was designed as a controlled, randomized and blinded efficacy study. Wherever possible, the principles of Good Clinical Practice (Veterinary International Conferenceon Harmonization (VICH)2000a ,b,c) and the WAAVP guideline for evaluating the efficacy of anthelmintics for dogs and cats (Jacobs et al.1994) were followed.Twenty-four purpose-bred domestic short-hair kittens(age 14  –  15 weeks) were acclimatized to the study facil-ity for 7 days. During the acclimatization period, threefaecal egg counts (FECs) were performed on consecu-tive days to demonstrate the absence of helminth infec-tions. The kittens had never received an anthelminthicor other drug that could have interfered with the studyobjective. Fitness for study inclusion was demonstrated by physical examinations prior to the experimental in-fection and prior to treatment. On study day 0, allkittens were inoculated with approximately 400 embry-onated T. cati eggs from a strain that had been main-tained in the lab since 2010 and was srcinally isolatedfrom a naturally infected cat in Germany.Based on sex and body weight, the kittens wererandomly allocated on study day 4 to three study groupswith eight cats each and a sex ratio of 1:1 (Table2).Treatments were performed on study day 5. Group 1 wastreated with the minimum therapeutic dosage of 3 mg/kgemodepside plus 12 mg/kg praziquantel (Profender®),corresponding to 0.12 ml/kg spot-on solution. The com-mercially available 0.35-ml pipette was emptied into a glass vial and the appropriate volume was withdrawnusing a 1,000- μ  l pipette and applied to the cat's skin at the base of the skull in front of the shoulder blades.Group 2 received the minimum therapeutic dosage of 2 mg/kg milbemycin oxime and 5 mg/kg praziquantel.The arithmetic mean weight of ten tablets of the batchwas calculated to determine the correct tablet mass per kilogramme. Excess tablet mass was rubbed off withsandpaper. The maximum discrepancy between target and actual tablet weight was +0.8 mg/kg, correspondingto +0.02 mg/kg milbemycin oxime. Group 3 was thenegative control group and was thus left untreated.During the prepatent period, the kittens were group-housed in their respective treatment group. Housing andgeneral maintenance was standardized across all ani-mals. The cats received a commercially available dryfood (Growth ™ , Royal Canin) and tap water ad libitum.General health observations were conducted daily duringcleaning and feeding activities and during social inter-action with the animal attendants. To monitor the pos-sible onset of patency, FECs were performed on pooledgroup samples three times per week starting on studyday 25. When the first pool of sample was positive, thecats were individually housed in cages and the number of eggs per gram of faeces was determined daily untilthe end of the study. Throughout the study, the rooms,cages and litter trays were carefully inspected to detect any spontaneously expelled worms. On study day 50,all cats were dewormed with a combination product of  praziquantel and pyrantel (Drontal®) according to themanufacturer's recommendations, and all faeces werecarefully searched for excreted worms for 3 days fol-lowing the treatment. Worms were counted and differ-entiated by stage and sex and total numbers were usedto calculate the product effect according to Abbot'sformula for controlled tests:Percentage of efficacy ð % Þ¼ 100 Â C  À T  ð Þ = C  where C  was the geometric mean of  T. cati in thecontrol group, and T  was the geometric mean of  T. cati in the treated group (Profender® or Milbemax®). Toconfirm the success of treatment, a final FEC was performed on study day 54 before the study was termi-nated. The experimental design is summarized inTable1.One-way analysis of variance was used to compare thegroups with regard to pre-treatment body weights. A non- parametric test (Mann  –  Whitney rank sum test) was used toanalyze worm count data and daily egg excretion. Results All cats stayed clinically healthy throughout the study peri-od. Treatment in groups 1 (Profender®) and 2 (Milbemax®)was tolerated well by all cats. Neither local nor systemiceffects were observed. There was no statistically significant  2124 Parasitol Res (2012) 111:2123  –  2127  difference between the groups regarding body weight onstudy day 4 (  p 0 0.954).On study day 36, the first positive faecal groupsample was obtained in group 3 (negative control),and from study day 42 until the end of the study, sevenout of eight cats in this group shed Toxocara eggs. Onecat stayed coproscopically negative throughout thestudy. In group 2 (Milbemax®), all cats were copros-copically positive from study day 41 onwards. In group1 (Profender®), seven out of eight cats stayed copros-copically negative throughout the study. One cat beganto shed Toxocara eggs on study day 42. Where eggexcretion was concerned, there was no statistically sig-nificant difference between group 2 (Milbemax®) andthe control group (  p ≥ 0.442). Egg counts in Profender®-treated animals differed significantly from the controlgroup from study day 41 until the end of the study(  p ≤ 0.035). After deworming at the end of the study, a total of 257 worms was collected in the negative controlgroup. Worms were recovered from all eight cats, andsix cats harboured an adequate infection ( ≥ 5 worms).The coproscopically negative cat harboured two maleworms and an immature female. In the Milbemax®-treated group, a total of 318 worms was collected.Worm numbers in this treatment group exceeded thenumbers in the control group; therefore, no treatment effect was evident. There was no statistically significant difference between the negative control group and theMilbemax®-treated group (  p 0 0.382). The treatment ingroup 1 (Profender®) was 98.5 % effective (  p <0.001).Only the coproscopically positive cat harboured sixworms (Table2). Spontaneously expelled worms werenot detected in any group. Individual worm counts are presented in Table3and the course of egg excretion is presented in graph form in Fig.1. The final FEC onstudy day 54 was negative in all cats, demonstrating thesuccess of treatment. Discussion The efficacy of the emodepside/praziquantel spot-on solu-tion against third-stage larva of  T. cati was comparable tothe level of control obtained in the studies for marketingauthorization (Reinemeyer et al.2005). Efficacy calcula-tions in the study presented here were based on worm countsafter deworming and not on worm counts determined at necropsy. Although desirable from an ethical point of view,this procedure has disadvantages that reduce the reliabilityof the results and is hence generally not acceptable inlaboratory studies for drug approval. Worms may be whollyor partially digested when passed in the faeces and maytherefore not be identified. This fact is of lesser importancefor  “ strong ” worms like ascarids, but can be critical for worms that are more fragile. Further unintended loss of worms may occur through coprophagia, mainly seen indogs, or ingestion of regurgitated worms as seen in dogsand cats. Finally, only a necropsy can prove that the animalwas completely cleared of an existing worm burden. As seenin this study, low worm burdens or infections with single-sex worms or immature worms cannot be detected by faecalexaminations. Despite these drawbacks, it can be concluded Table 1 Summary experimental design Study day Activity − 7 to − 1 Acclimatization, FEC over 3 consecutive days0 Experimental infection with 400 T. cati eggs4 Randomization and allocation to groups5 Treatment in groups 1 and 225  —  onset of positivegroup samplesHousing in groups, FEC three times a week ingroups until positive, then individual housing49  —  onset of positivegroup samplesFEC in individually housed cats50 Anthelmintic treatment 51  –  53 Collection of worms in faeces54 FEC Table 2 Group details SD study day, F  female,  M  male Group Cat no.Sex Body weight on SD 41. Profender® 1 F 1.752 F 1.653 F 1.604 F 1.205 M 2.156 M 2.007 M 1.908 M 1.452. Milbemax® 1 F 1.652 F 1.603 F 1.604 F 1.305 M 2.156 M 2.107 M 1.908 M 1.353. Untreatedcontrol1 F 1.802 F 1.603 F 1.554 F 1.455 M 2.306 M 2.107 M 1.708 M 1.50 Parasitol Res (2012) 111:2123  –  2127 2125  that reliable data were generated in this study. Resultsobtained in group 1 (Profender®) were similar to former studies and the negative control group showed an ade-quate level of infection. As is often seen in experimen-tally infected animals, the individual worm burdens inthe negative control group were highly variable (range3  –  112), but the requirement for six adequately infectedanimals was fulfilled. No efficacy could be observed in the milbemycin oxime-treated cats. Numbers of excreted worms and eggs passed inthe faeces exceeded those in the control group. Treatment was performed 5 days after the experimental infection tocoincide with the migrating stage of the parasite (VICH2000c). Although information on the detailed migratory behaviour of  T. cati is scarce, observations by Sprent (1956) showed that after egg infection, the larvae performa liver   –  lung migration and can be found in the lungs at thistime point of infection. To be effective against these early-migrating larvae, an active ingredient has to be present at a sufficient level within these tissues and/or the blood streamat a certain time point. According to Reinemeyer and Court-ney (2001), 5  –  10 % of an orally administered dose of milbemycin oxime is absorbed; the major part is excretedwith the faeces. Although milbemycin oxime in the formu-lated tablet Milbemax® is effective against migrating larvalstages of  D. immitis in the cat (Genchi et al.2004), theresults obtained under the conditions of this study lead tothe assumption that migrating T. cati larvae are not likewise affected, either due to low drug levels or to anunfavourable tissue distribution with regard to this para-site. It can be concluded that to date, the spot-on formu-lation of emodepside and praziquantel remains the onlyanthelmintic with proven efficacy against migrating T.cati larvae. Table 3 Individual wormcounts Group Cat no. No. of female T. cati  No. of male T. cati  No. of  preadultsTotal no.of  T. cati Geometric meanworm count 1. Profender® 1 0 0 0 0 0.32 4 2 0 63 0 0 0 04 0 0 0 05 0 0 0 06 0 0 0 07 0 0 0 08 0 0 0 02. Milbemax® 1 5 2 0 7 27.72 6 5 0 113 10 22 10 424 9 30 7 465 9 13 0 226 55 53 27 1357 11 7 2 208 17 18 0 353. Untreated control 1 5 6 0 11 18.12 7 18 0 253 1 2 0 34 1 3 0 45 8 6 4 186 50 54 8 1127 14 27 24 658 5 14 0 19 40 41 42 43 44 45 46 47 48 490200040006000 ProfenderMilbemaxControl Days post infection    M  e  a  n   E   P   G Fig. 1 Arithmetic mean faecal egg count and standard deviation on theindividual study days2126 Parasitol Res (2012) 111:2123  –  2127
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