Efficacy of a novel topical combination of fipronil, (S ... - Core

Veterinary Parasitology 202 (2014) 64–68

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Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against larval and adult stages of the cat lungworm, Aelurostrongylus abstrusus Martin Knaus a,∗ , S. Theodore Chester b , Joseph Rosentel b , Axel Kühnert a , Steffen Rehbein a a b

Merial GmbH, Kathrinenhof Research Center, 83101 Rohrdorf, Germany Merial Limited, Duluth, GA 30096, USA

a r t i c l e

i n f o

Keywords: Aelurostrongylus abstrusus Topical Fipronil (S)-methoprene Eprinomectin Praziquantel Efficacy Lungworm Cat

a b s t r a c t The efficacy of a novel topical combination of fipronil 8.3% w/v, (S)-methoprene 10% w/v, eprinomectin 0.4% w/v, and praziquantel 8.3% w/v (BROADLINE® ,1 Merial) against larval and adult Aelurostrongylus abstrusus lungworms in cats was assessed in a controlled laboratory study. The study included 48 purpose-bred, short-haired cats which were each inoculated with 225 infective A. abstrusus larvae. The cats were formed into eight blocks based on pre-treatment bodyweight and were then, within each block, randomly allocated to one of six treatment groups: untreated control; treated once when A. abstrusus were expected to be third-stage larvae (4 days post inoculation [dpi]), fourth-stage larvae (7 dpi), immature adults (14 dpi) or adult nematodes (32 dpi), or treated twice, once when A. abstrusus were expected to be third-stage larval and once again when A. abstrusus were expected to be adult nematodes (4 dpi + 32 dpi). Cats weighing ≥0.8–2.5 kg received one 0.3 mL applicator and cats weighing >2.5–7.5 kg received one 0.9 mL applicator. For determination of the efficacy of treatments, lungworm larval counts were established on faecal samples collected from all cats 32, 39, 46, 53 and 60 dpi. At each occasion from 46 dpi on, cats treated with fipronil, (S)-methoprene, eprinomectin and praziquantel had significantly lower A. abstrusus larval counts than the untreated controls with percentage reductions of 91.6% (cats treated 14 dpi; P = 0.012), ≥98.9% (cats treated either 4 dpi, 7 dpi or 32 dpi; P < 0.001) or >99.9% (cats treated 4 dpi + 32 dpi; P < 0.001) at 60 dpi. Thus, the novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel was highly effective in the prevention and treatment of A. abstrusus lungworm infection in cats. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

∗ Corresponding author. Tel.: +49 8032 70750; fax: +49 8032 707525. E-mail address: [email protected] (M. Knaus). 1 Broadline® is a trademark of Merial; all other marks are the property of their respective owners.

Parasitic nematode infections of the respiratory tract of both dogs and cats have received increasing attention by both clinicians and parasitologists in the recent years and prompted specific surveys on feline parasitic lungworm infections. The metastrongyloid Aelurostrongylus abstrusus

http://dx.doi.org/10.1016/j.vetpar.2014.02.042 0304-4017/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/3.0/).

M. Knaus et al. / Veterinary Parasitology 202 (2014) 64–68

is apparently the most prevalent pulmonary parasite of domestic cats (reviewed by Traversa et al., 2010; Mircean et al., 2010; Di Cesare et al., 2011; Knaus et al., 2011; Ramos et al., 2012; Barutzki and Schaper, 2013; Capári et al., 2013; Riggio et al., 2013; Spada et al., 2013; Waap et al., 2013) while other lungworms were reported less frequently – Capillaria aerophila (Mircean et al., 2010; Knaus et al., 2011; Di Cesare et al., 2011; Mugnaini et al., 2012) or occasionally only – Oslerus rostratus (Juste et al., 1992) and Troglostrongylus spp. (Jefferies et al., 2010; Brianti et al., 2012). After the original description of A. abstrusus isolated from a cat in Germany in 1890 (Mueller, 1891), the global distribution of this parasite in felines was documented through numerous case reports and epidemiological surveys (Bowman et al., 2002). Aelurostrongylus abstrusus resides in the lower respiratory tract, particularly in the bronchioli and alveoli (lung parenchyma) of felines. Female nematodes are oviparous and eggs that are laid develop into firststage larvae in the alveoli and alveolar ducts of the lungs. First-stage larvae pass through the respiratory into the gastrointestinal tract and are shed with the faeces. Aelurostrongylus abstrusus develops to the infective thirdstage larvae in a wide spectrum of terrestrial snail and slug species which serve as intermediate hosts in the life cycle of this parasite. However, cats probably acquire the parasite under natural conditions through ingestion of prey (e.g., small mammals, birds, reptiles, or amphibians) which feed on gastropods and serve as paratenic hosts. In cats, larvae penetrate the upper gastrointestinal tract within the first day of infection and reach the lungs shortly thereafter. After two more moults, females begin to lay eggs in the fourth week following infection (Anderson, 2000; Bowman et al., 2002; Grewal et al., 2003). Many naturally acquired A. abstrusus infections are apparently asymptomatic or subclinical in nature. Several authors, however, have reported a range of respiratory signs associated with Aelurostrongylus infections in cats (e.g., Scott, 1973; Losonsky et al., 1978; Grandi et al., 2005; Payo-Puente et al., 2005; Iannino et al., 2013), and necropsy and sophisticated intravital diagnostic techniques revealed pathological changes of varying degree (e.g., Mackenzie, 1960; Stockdale, 1970; Payo-Puente et al., 2005; Dennler et al., 2013). Studies have shown that cats presenting signs of respiratory disease tested positive more frequently for A. abstrusus than cats of unknown clinical status (Barutzki and Schaper, 2011, 2013). Conditions compromising the immune system of cats are discussed to play a role in the clinical manifestation of the infection and may complicate diagnosis (Mackenzie, 1960; Hamilton, 1963; Schuster et al., 1999; Tüzer et al., 2002; Grandi et al., 2005; Traversa et al., 2008; Dürr, 2009). The study reported here was conducted to evaluate the efficacy of a novel topical combination of fipronil, (S)methoprene, eprinomectin and praziquantel against larval and adult stages of A. abstrusus in cats using an induced infection design.

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2. Material and methods The design of the study was in accordance with the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) – GL7, “Efficacy of Anthelmintics: General Requirements” (Vercruysse et al., 2001) and GL20 “Efficacy of Anthelmintics: Specific Recommendations for Felines” (Vercruysse et al., 2002), and the “World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anthelmintics for dogs and cats” (Jacobs et al., 1994). Recovery of nematodes associated with the lungs of cats has been considered impracticable and hence, as recommended by WAAVP, monitoring of faecal lungworm larval counts in treated and untreated animals was used to assess the efficacy against A. abstrusus (Jacobs et al., 1994). The study was conducted in compliance with VICH GL9, entitled Good Clinical Practice and in compliance with local animal welfare legislation and was approved by an Independent Animal Care and Use Committee. All personnel involved in collecting efficacy data were blinded to the treatment assignment of the animals. 2.1. Experimental animals and inoculation The study included 48 purpose-bred short-haired cats. Cats weighed 2.0–4.7 kg prior to treatment and were approximately 7–14 months old (Table 1). The animals were housed individually throughout the study under identical conditions. All cats were negative for A. abstrusus larvae prior to inoculation as confirmed by examination of faeces subjected to the Baermann-Wetzel larval migration technique. Each cat was inoculated orally once with approximately 225 infective third-stage larvae which were obtained from experimentally infected Helix aspersa snails. For inoculation, a recent field isolate (as defined per VICH GL 7, Table 1 Characteristics of experimental cats used in the study of the efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against larval and adult stages of the cat lungworm, Aelurostrongylus abstrusus. Treatment groups

Control, untreated Treatedb , 4 dpic , d Treated, 10 dpie Treated, 14 dpif Treated, 32 dpig Treated, 4d dpi + 32g dpi a

Animal data Age (months)

Sexa

7–11 7–10 7–10 7–10 7–14 7–10

3 M, 5 F 4 M, 4 F 4 M, 4 F 5 M, 3 F 5 M, 3 F 3 M, 5 F

Pre-treatment bodyweight (kg) 2.19–4.64 2.24–4.26 2.00–4.69 2.01–4.41 2.20–4.44 2.19–4.36

M = male, F = female. Broadline® = fipronil (8.3% w/v), (S)-methoprene (10% w/v), eprinomectin (0.4% w/v), and praziquantel (8.3% w/v); 0.3 mL, cats ≥0.8–2.5 kg body weight; 0.9 mL, cats >2.5–7.5 kg body weight. c dpi = days post inoculation. d Aelurostrongylus abstrusus expected to be third-stage larvae. e Aelurostrongylus abstrusus expected to be fourth-stage larvae. f Aelurostrongylus abstrusus expected to be immature adults. g Aelurostrongylus abstrusus expected to be adults. b

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Vercruysse et al., 2001) from a naturally infected cat from Albania was used. The isolate was passed through H. aspersa and cats for approximately four years in the laboratory before being used in this study. 2.2. Study design The study utilized a randomized block design based on pre-treatment bodyweight. Cats were ranked based on decreasing bodyweight and blocks of six cats each were formed. Within blocks, cats were allocated randomly to one of six groups: untreated (control); treated once when A. abstrusus were expected to be third-stage larvae (4 days post inoculation, dpi); treated once when A. abstrusus were expected to be fourth-stage larvae (7 dpi); treated once when A. abstrusus were expected to be immature adults (14 dpi); treated once when A. abstrusus were expected to be adults (32 dpi); treated twice, once when A. abstrusus were expected to be third-stage larvae and once again when they were expected to be adults (4 dpi + 32 dpi). Treatment times were selected based on the developmental cycle of A. abstrusus in the cat (reviewed by Anderson, 2000). The treatment, fipronil 8.3% w/v, (S)-methoprene 10% w/v, eprinomectin 0.4% w/v, praziquantel 8.3% w/v, (Broadline® , Merial), was administered once topically directly on the skin in the midline of the neck between the base of the skull and the shoulder blades in a single spot. Cats weighing ≥0.8–2.5 kg received one 0.3 mL applicator, and cats of >2.5–7.5 kg body weight received one 0.9 mL applicator. The dosage corresponded to the label dose of the novel combination formulation. All cats were observed hourly for four hours post-treatment and thereafter once daily until end of the study for health problems or adverse events. 2.3. Faecal examinations Faecal samples were collected from all cats on 32 dpi, and weekly thereafter until 60 dpi and examined for A. abstrusus larvae (Fig. 1) using the quantitative BaermannWetzel migration technique utilizing individual 10 g faecal samples. 2.4. Data analysis Aelurostrongylus abstrusus larval counts were transformed to the natural logarithm of (count +1) for calculation of geometric means for each treatment group and time point. Efficacy for each treated group and time point were determined by calculating the percent efficacy as 100[(C − T)/C], where C was the geometric mean among untreated controls and T was the geometric mean among the treated animals. The log-counts of the treated group was compared to the log-counts of the untreated control group. In addition, pairwise comparisons among the treated groups were conducted at each study day. Both sets of comparisons used an F-test adjusted for the allocation blocks used to randomize the animals to the treatment groups. The mixed procedure in SAS® Version 9.1.3 was used for the analysis, with the treatment groups listed as

Figure 1. Aelurostrongylus abstrusus larva 1 extracted from a Baermann. ©Merial.

a fixed effect, and the allocation blocks listed as a random effect. The comparisons of the treated group with the untreated controls were tested using a two-sided at the significance level ˛ = 0.05. For pairwise comparisons among the treated groups, to adjust for the a posteriori multiple comparisons, the Bonferroni adjustment was used: within each time point, two treated groups were significantly different if P < 0.05/10 = 0.005. 3. Results No adverse experiences or other health problems were observed after treatment application or throughout the course of the study, indicating that the treatment was well accepted. All cats in this study were considered as normal based on daily clinical observations; no clinical signs indicating respiratory distress were observed in any of the cats. The results of the study as regards A. abstrusus larval counts and percentage efficacy are summarized in Table 2. The study was considered valid in the sense of the adequacy-of-infection criterion per VICH GL7 and VICH GL20 as at least seven cats in the control group shed A. abstrusus larvae throughout 39–60 dpi. No A. abstrusus larvae were recovered from the faeces of the cats collected 32 dpi. Faecal examination at 39 dpi revealed A. abstrusus larvae in the faeces of seven untreated control cats and the cats treated on 32 dpi. As female A. abstrusus were observed to begin laying of eggs as early as 25 dpi (Stockdale, 1970), this finding indicates that the larvae recovered from the cats’ faeces developed from eggs produced already before 32 dpi. Aelurostrongylus abstrusus larval counts from 46 dpi, 53 dpi and 60 dpi demonstrated ≥98.9%, ≥99.3%, ≥91.6% and ≥99.6% efficacy (P ≤ 0.012) of a single treatment with Broadline® spot on against third-stage larvae, fourthstage larvae, immature adults or adults of A. abstrusus, respectively; two consecutive treatments one month apart


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Table 2 Larval counts and therapeutic efficacy of the topical combination product against different developmental stages of Aelurostrongylus abstrusus in cats. Geometric meana A. abstrusus larval counts per gram of faeces (range) 39 dpi Control, untreated Treatedc , 4 dpid , e Treated, 10 dpif Treated, 14 dpig Treated, 32 dpih Treated, 4 dpie + 32 dpih

33.8a (0–384) 0b (0) 0b (0) 0b (0) 44.0a (1.1–1690) 0b (0)

46 dpi 309.1a (0.2–1440) 0.6b (0–4.4) 0.9b (0–10.3) 0.4b (0–1.9) 0.8b (0–8.3) 99.9% reduction of larval shedding throughout (P < 0.001). For each time point, there was no statistically significant difference of the lungworm larval counts among the groups treated apart from one occasion: Aelurostrongylus abstrusus larval counts of the cats treated 14 dpi were higher than those of the cats which received two treatments one month apart (4 dpi + 32 dpi; P = 0.002) at 60 dpi. 4. Discussion This is the first study demonstrating a high efficacy of the novel topical combination formulation containing the avermectin eprinomectin against larval and adult stages of A. abstrusus in cats. To compensate for irregular larval release and shedding and the potential limited sensitivity of the Baermann-Wetzel technique, faecal examinations in this study were conducted in weekly intervals for three weeks following first isolation of A. abstrusus larvae 39 dpi, thus allowing for a better understanding on the sustainability of the treatment with the novel combination formulation. The results of this study with time points of treatment selected based on the developmental cycle of A. abstrusus (reviewed by Anderson, 2000) indicate that the administration of the combination product to infected cats significantly affected all parasitic stages in the definitive host and prevented the establishment of A. abstrusus to a very high level. Efficacy against pre-patent developmental stages is an important feature of anthelmintics as this property contributes to a more sustainable control of aelurostrongylosis in cats, and it can be assumed that the pulmonary changes, which are progressing during the infection A. abstrusus (cf. Stockdale, 1970; Dennler et al., 2013), will be markedly reduced. This is the first reported controlled laboratory study in which a macrocyclic lactone demonstrated efficacy against all parasitic stages of A. abstrusus in the cat. Macrocyclic lactone compounds have been used as anthelmintics in pets for several years and were shown to provide a high efficacy against infections of various nematode parasites

(reviewed by Nolan and Lok, 2012). In cats, the use of various macrocyclic lactones administered by different routes and at different doses against naturally acquired A. abstrusus infections were reported previously in several case reports with inconsistent treatment efficacies (Blagburn et al., 1987; Kirkpatrick and Megella, 1987; Tüzer et al., 2002; Reinhardt et al., 2004; Grandi et al., 2005) or studies conducted under field conditions lacking negative control animals (Brianti et al., 2008; Traversa et al., 2009a,b; Iannino et al., 2013). As seen previously in field studies (Brianti et al., 2008; Iannino et al., 2013), a complete reduction of A. abstrusus larval shedding following a single treatment with macrocyclic lactone products is typically not achieved, and repeated treatments along with repeated faecal examinations to monitor the treatment effect may be necessary. Monthly use of Broadline® is expected to provide a very high level of efficacy, as demonstrated by the results in cats treated on post-infection days 4 and 32. Conflict of interest The work reported herein was funded by Merial Limited, GA, USA. All authors are current employees of Merial. Acknowledgments The authors gratefully acknowledge the technical assistance of Heike Ryl and Martin Visser. References Anderson, R.C., 2000. Nematode Parasites of Vertebrates. Their Development and Transmission, 2nd ed. CABI Publishing, Wallingford, Oxon, UK. Barutzki, D., Schaper, R., 2011. Results of parasitological examinations of faecal samples from cats and dogs in Germany between 2003 and 2010. Parasitol. Res. 109 (Suppl.), S45–S60. Barutzki, D., Schaper, R., 2013. Occurrence and regional distribution of Aelurostrongylus abstrusus in cats in Germany. Parasitol. Res. 112, 855–861. Blagburn, B.L., Hendrix, C.M., Lindsay, D.S., Vaughan, J.L., 1987. Anthelmintic efficacy of ivermectin in naturally parasitized cats. Am. J. Vet. Res. 48, 670–672.

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