Dynamics of distribution and efficacy of different spot-on permethrin formulations in dogs artificially infested with Dermacentor reticulatus

<p>Abstract</p> <p>Background</p> <p>Varying reports concerning the duration and reliability of different permethrin preparations' efficacy can be found in the literature. The aim of this study was to investigate the dynamics of the distribution and efficacy of four different spot-on formulations with permethrin as the active ingredient formulated with different solvents. To examine the influence of these solvents on the speed of distribution and the acaricidal activity of permethrin in the coat, an <it>in vivo </it>study under laboratory conditions was performed. Six dogs per test period were treated with the recommended dose and 1, 14 and 28 days after treatment dogs were infested with <it>Dermacentor reticulatus </it>ticks: a) on the back, near the application site, and b) on the hind leg, the greatest possible distance from the application site. Efficacies were determined 6 hours after tick infestation to examine the repellent effect and the speed of kill of the products which plays an important role in the context of tick transmitted diseases.</p> <p>Results</p> <p>After six hours of exposure, a significant acaricidal efficacy (p < 0.001) could be observed in all treated groups over the whole duration of the study, regardless of which product was used. While the arithmetic mean of attached ticks was < 3 on Day 1, numbers increased over the course of the study to a mean of > 9 on Day 28. However, most of these ticks were dead even 28 days after treatment, as the mean of live attached ticks was still < 2. Significant differences could neither be found between the different permethrin spot-on formulations, nor between the two parts of the body (p > 0.05).</p> <p>Conclusions</p> <p>All products were able to kill ticks within six hours following infestation from Day 1 to Day 28 after treatment. Additionally, no significant difference between the tick numbers on the back and the hind leg could be found at any time, which implies a homogenous distribution of permethrin over the body. The efficacy of all four products was comparable during the whole study period, showing that the different solvents do not significantly affect the dynamics of distribution.</p

Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections

Angiostrongylus vasorum is an increasingly reported parasite in Europe that develops in dogs after ingestion of infective third stage larvae (L3) that reside in gastropod molluscs which are needed to complete the parasite's life-cycle. Infection can produce a diversity of clinical signs, determined by involvement of the respiratory, neurological, and/or coagulation system, with a likely fatal outcome in the absence of treatment. Few drugs have been shown to reliably prevent infection, and data on treatment of infections is limited. A controlled, randomized, partially blinded laboratory study was therefore executed to evaluate the efficacy and safety of a combination tablet of spinosad/milbemycin oxime in dogs inoculated with approximately 250 A. vasorum L3. Sixteen healthy nematode free adult dogs were randomly allocated to two study groups of 8 dogs each. Thirty days post inoculation (dpi) all dogs in the fed state were treated: dogs in group B were treated with spinosad and milbemycin oxime at the dose rates of 45-60 mg/kg and 0.75-1.0mg/kg bodyweight, respectively, approximately the lower half portion of the expected full unit dose range; dogs in group A were treated with placebo tablets. All dogs were euthanized and necropsied 56-58 dpi. The heart and lungs were examined to determine the presence of A. vasorum. All placebo group dogs were infected at necropsy with counts ranging from 22 to 98 adult worms and a geometric mean worm count of 55.2. In contrast, the geometric mean worm count in the spinosad/milbemycin oxime group was 0.7 with worm numbers ranging from 0 to 8. The results of this study demonstrate that a single treatment with the tablet combination of spinosad and milbemycin oxime administered 30 dpi provided 98.8% preventive efficacy against development of adult A. vasorum infections. Monthly treatments with spinosad and milbemycin oxime have the potential to prevent the establishment of infections with A. vasorum in dogs

Efficacy of Emodepside/Praziquantel spot-on (Profender®) against adult Aelurostrongylus abstrusus Nematodes in experimentally infected cats

The adulticidal efficacy of a topical combination of emodepside 2.1 % (w/v) plus praziquantel 8.6 % (w/v) (Profender® spot-on for cats, Bayer) against adult Aelurostrongylus abstrusus nematodes was evaluated in two randomised, placebo-controlled laboratory efficacy studies. Each study involved 16 cats experimentally inoculated with L3 (800 and 600 each in studies no. 1 and 2, respectively) and randomised into two study groups of 8 cats each after onset of patency. While cats in the treatment group in study no. 1 received a single spot-on application at the minimum therapeutic dose (3 mg/kg emodepside and 12 mg/kg praziquantel), cats in study no. 2 were treated twice with an interval of 14 days. The faecal output of first stage larvae was monitored throughout the study. Necropsy was conducted 4 or 5 weeks after the (first) treatment and the worm counts were used for efficacy calculations. The control groups showed a geometric mean of the total worm count (live and dead worms) of 28.8 (study no. 1) and 17.6 (study no. 2), respectively. All control animals were infected. While the single treatment in study no. 1 resulted in a reduction of the total worm burden by 73.0 % (p = 0.0070), the treatment protocol in study no. 2 was 99.2 % effective (p = 0.0035). Based on live worm counts, the efficacy in study no. 2 was 100 % (p = 0.0030). It is concluded that two applications of Profender® spot-on given two weeks apart represent a safe and highly efficacious treatment regime against feline aelurostrongylosis

Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM

Parasite species identification by melting curve analysis.

<p>Fecal extracts from samples containing <i>T. canis</i>, <i>T. cati</i> or a mixture of both species were used as template for real-time PCR in the presence of EvaGreen followed by a high resolution melting curve analysis. Melting peaks obtained from maxima in the plot of the first deviation of the fluorescence intensity d(RFU)/dT were 88.4–88.5°C for <i>T. cati</i> and 90.3–90.4°C for <i>T. canis</i> (a). Amplicons from the mixed samples showed both peaks. Melting curves were normalized (b) and a difference plot (c) was calculated by subtracting the mean of both <i>T. canis</i> samples from all individual curves. Results for one of three representative experiments are shown.</p

Species determination for human hookworm samples.

<p>(A) Eight human and one canine (<i>A. caninum</i>) sample (+) together with hookworm negative fecal extract (−) were analyzed in duplicate using the ITS-2 primer pair. <i>N. am.</i>, <i>Necator americanus</i>; <i>A. du.</i>, <i>A. duodenale</i>; M, marker (100 bp ladder, Fermentas). (B, C) Representative PCR products were purified and 150 ng were digested with <i>Rsa</i>I and separated using the DNA1000 LabChip® which has a sizing range from 25–1000 bp. Theoretical fragment sizes for <i>N. americanus</i> are 313, 53, 40 and 11 bp and 159, 139, 35, 28, 28, 16 and 12 bp. In silico digestion of <i>A. duodenale</i> or <i>A. caninum</i> fragments leads to identical fragment sizes of 198 and 113 bp. The gel view is shown in (B) for five samples and the electropherogram in (C) for two samples.</p

Flow chart comparing different egg purification protocols.

<p>Steps that were used in all protocols are shown in yellow, variant purification protocol steps in either orange or red. The latter color indicates mandatory steps for successful amplification in the final protocol in method C. The fourth protocol was modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061285#pone.0061285-Bott1" target="_blank">[14]</a> by increasing the amount of feces to 10 g to allow comparison with the other approaches.</p

Determination of amplification efficacy by real-time PCR.

<p>Fourfold serial dilutions of fecal sample extract from a <i>T. cati</i> infected cat (1∶4, dilution factor 0.25 to 1∶1024, dilution factor 0.00098) and tenfold serial dilutions of plasmid DNA (10⁶ to 10¹ copies) were used as template for real-time PCR in the presence of EvaGreen fluorescence dye. (A) Amplification plots showing signal accumulation measured in relative fluorescence units (RFU) with increasing cycle number. (b) Regression curves for fecal samples (red) and plasmid DNA (blue) were calculated with GraphPad Prism 5. Goodness of fit in terms of R² and slopes (with 95% confidence intervals) are given and slopes were used to calculate PCR efficiencies (Eff). Both regressions curves are virtually parallel and no significant difference between slopes could be found. (c) Amplification efficacy was also calculated from the slopes of individual amplification plots with LinRegPCR. Individual efficacies for all fecal (dots) and all plasmid samples (squares) as well as means ± SD are presented. A Student’s t test was used to compare PCR efficiencies between both groups but no significant differences could be detected.</p